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

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Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors Joost J. Oppenheim1,*, O. M. Zack Howard2 and Edward Goetzl3 1

Laboratory of Molecular Immunoregulation, Intramural Research Support Program, Building 560, Room 21-89A, Frederick, MA 21702-1201, USA 2 Frederick Cancer Research and Development Center, Frederick, MA 21702-1201, USA 3 University of California San Francisco, U-B8B, 533 Parnassus, Box 0711, San Francisco, CA 94143-0711, USA * corresponding author tel: 301-846-1551, fax: 301-846-7042, e-mail: [email protected] DOI: 10.1006/rwcy.2000.02011.

SUMMARY This introductory review serves to introduce the reader to several families of cellular chemoattractants including cytokines known as chemokines, as neuropeptides, components of the complement cascade, and cyclooxygenase pathways. These ligands share the capacity to utilize seven transmembrane (STM) G protein-coupled receptors. As is true of other cytokines, these ligands are multifunctional and, in addition to mobilizing inflammatory cells to sites of infection and injury, act on a wide spectrum of progenitor cells and on nonleukocytes. Consequently, some of these ligands participate in fetal development, others on hematopoiesis on trafficking and homing of cells to tissues and organs, on the induction of inflammatory and immune responses, and on regulating cell adhesion and angiogenesis. We will also review reports that numerous viruses and microorganisms

have adapted and learned to circumvent these mediators of host defense by producing agonists or antagonists or, as illustrated by HIV-1, by using chemokine receptors as entry sites for cellular invasion.

INTRODUCTION Well over a century ago histopathological studies revealed inflammatory sites to be heavily infiltrated by leukocytes (as reviewed by Gallin et al., 1992). Phagocytic leukocytes present at sites of infection engulfed bacteria. Metchnikoff was the first to recognize that phagocytes engage in host defense by diapedesis from the circulation into inflamed tissues, and by internalizing and digesting the microorganisms (Metchnikoff, 1901). This raised questions as to 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 or to endogenous mediators. We now know that both contribute. Although scientists tried various in vitro approaches, it was not until the invention of the Boyden chamber in the 1960s that it became possible quantitatively to assay the directional in vitro migration of cells through intervening filters in response to a chemotactic attractant (Boyden, 1962). This led to the identification of potent exogenous and endogenous chemotactic factors for phagocytic cells such as fMLP fragments of bacterial cell walls, C5a generated by activation of the complement cascade, cell-derived lipid moieties such as platelet-activating factor (PAF), and leukotriene B4 (LTB4) generated by arachidonic acid metabolism. At the end of the 1960s conditioned media of antigenically activated T cell cultures were shown to contain lymphokines that were chemotactic for monocytes (Ward et al., 1969) and neutrophils (Ward et al., 1970). During the next 30 years, a wide variety of peptides derived from bacteria, serum proteins, and various cell types and tissues have been shown to have the capacity to direct cell migration along gradients of increasing concentration to their site of origin. Furthermore, virtually all cell types, including T and B lymphocytes, natural killer (NK) cells, eosinophils, basophils, dendritic cells (DC), and nonleukocytic cells, such as endothelial cells, fibroblasts, smooth muscle cells, have the capacity to respond to chemotactic stimuli. Although it is difficult to demonstrate that cells migrate along a gradient in vivo, in vitro assays of chemotaxis in general reflect in vivo inflammatory or cell-homing responses and are therefore regarded as correlates of cellular immunity. Even though widely different types of chemoattractants have been identified, they have a similar signaling mechanism, bind to specific members of the family STM receptors and activate Gi-type G proteins. These receptors are characterized by biphasic dose responses and their capacity to be rapidly turned on and off. At lower ligand concentrations, STM receptor engagement rapidly induces signal transduction, resulting in changes in cell shape with pseudopod formation at the leading edge, actin polymerization, redistribution and activation of cell surface adhesion proteins leading to adhesion to endothelial cells, diapedesis through the endothelium, and migration along a gradient of increasing chemoattractant concentration present on cell surfaces and matrix proteins to the site of origin of the chemoattractant. At increased concentrations of about a log or more, the chemoattractants induce cells to degranulate with release of enzymes, histamine, antimicrobial peptides and the production of oxygen radicals, all of which have potential antimicrobial effects. At even higher

concentrations these responses will be attenuated and the cells become desensitized to subsequent stimulation by the ligand. This is associated with transient receptor phosphorylation, internalization, and ligand degradation, and can be reversed by recycling and reexpression of the receptor on the cell surface. A wide variety of ligands that use STM receptors and share signal transduction pathways will be considered and compared in this overview. This includes cell-derived chemokines, LTB4, PAF, complementderived C5a, bacterial-derived fMLP, neuropeptides, and related ligands. The contribution of the individual ligands and their receptors to host defense will be discussed in greater detail in the individual chapters. In order to minimize redundancy, this overview will provide tables and figures aimed at comparing the properties of these ligands and receptors to one another and will consider them in groups based on their shared pathophysiological roles. We will begin by discussing chemokines, followed by a brief introductory discussion of other chemotactic ligands that utilize STM.

CHEMOKINES: HISTORICAL DISCOVERY Based on the fact that interleukin-1 (IL-1) induces migration of leukocytes to sites of injection, IL-1 was thought to have chemotactic activity. In fact, leukocyte-derived chemoattractants for phagocytic cells were present in partially purified preparations of IL-1 (Luger et al., 1983; Sauder et al., 1984). However, when purified to homogeneity, IL-1 lost its chemotactic activity. Further studies revealed that the partially purified IL-1 preparations were contaminated with both a chemotactic factor for monocytes and a monocyte-derived neutrophil chemotactic factor, termed MDNCF (Yoshimura et al., 1987, 1989; Matsushima et al., 1988, 1989). These paradoxical observations were resolved by data showing that IL-1 was a potent inducer of these chemotactic factors. The same mononuclear cell-derived neutrophil attractants were also identified by several other laboratories of (Schroder et al., 1987; Walz et al., 1987). Later, Larsen et al. (1989) reported that MDNCF was chemotactic for T lymphocytes as well as neutrophils, and, based on the presumed involvement of MDNCF in the immune response, it was renamed IL-8 (Larsen et al., 1989; Oppenheim et al., 1991). Analysis of DNA sequence data revealed that IL-8 was a member of a cytokine gene family with related structural features. IL-8 was the first member of this

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors group to be shown to have substantial chemotactic activity and this rapidly stimulated investigators to identify other family members with chemotactic effects on various cell types. The first international meeting focusing on this group of chemoattractant cytokines was organized by John Westwick in 1988 at the Royal College of Surgeons in London, UK (Westwick et al., 1989). Enormous progress has been made, resulting in the identification of over 50 ligands to date, using techniques embodied in bioinformatics, differential nucleic acid and protein screens and protein purification. Although the naming of the individual molecules has been based on the rather anarchistic proclivities of investigators, the catchy term chemokine (short for chemotactic cytokines) was proposed by the Third International Symposium on Chemotactic Cytokines held in Baden, Austria in 1992 (Kunkel et al., 1995). The terminology for the STM G proteincoupled receptors for chemokines has been simplified and they are termed CXCR1-5, CCR1-8, XCR1, or CX3CR1 depending on structural features (as will be discussed) of the subfamily of the binding ligand (as proposed at the Second Gordon Conference on Chemotactic Cytokines held at Plymouth, NH in 1996).

OVERVIEW OF CHEMOKINE PATHOPHYSIOLOGY Like most other cytokines, chemokines are multifunctional. In addition to mobilizing inflammatory cells to sites of infections and injury, they also engage in regulation of cell adhesion, angiogenesis, fetal development, hematopoiesis, immune responses, and the trafficking and homing of lymphocytes and dendritic cells to their appropriate locations in lymphoid tissues (reviewed in Baggiolini et al., 1994, 1997; BenBaruch et al., 1995; Kunkel et al., 1995; Oppenheim et al., 1996; Baggiolini, 1998; Krakauer et al., 1999; Zlotnik et al., 1999). In addition to their afore-mentioned beneficial effects, chemokines can participate in deleterious reactions and at times are pre-empted by pathogens to the detriment of the host. Chemokines may also have undesirable injurious side-effects by contributing to autoimmune diseases and participate in reperfusion injury. In addition, certain microbial organisms, including Plasmodium vivax and HIV-1, have been found to exploit chemokine receptors as sites for host cell entry (Fauci, 1996). Furthermore, a number of viral gene products are homologous to the chemokines or chemokine receptors and

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presumably are used to circumvent host defenses (Murphy, 1996, 1997).

CHEMOKINE CLASSIFICATION The chemokine subfamilies are delineated by the number of cysteines and presence of intervening amino acids, leading to common designations of CXC (or ), CC ( ), C ( ), and CX3C (). All but two of the chemokines belong to the CXC and CC subgroups, and there is only one each of the C and CX3C chemokines. The linear amino acid alignment of the human chemokine subfamilies is shown in Figure 1. However, within a subfamily the homology can range from 25% to 95%. Intramolecular cysteine bonds are formed between C1 and C3, C2 and C4, of the CXC and CC subfamilies, resulting in stably folded molecules. A subgroup of the CC chemokines consisting of I-309, HCC-2, MPIF1, and SLC, have six cysteines (Miller et al., 1990; Schulz-Knappe et al., 1996; Nagira et al., 1997; Youn et al., 1997, 1998). In I-309, HCC-2, and MPIF1 one additional cysteine is located within the sheet domain and a second one is located in the C-terminal  helix, suggesting that these cysteines may form a third disulfide bond anchoring the C-terminal helix to the sheet. SLC has two cysteines on the C-terminal domain. Eotaxin 2 has an unpaired fifth cysteine at the C-terminus (Nagira et al., 1997). The effect of these additional cysteines on the secondary structure and on the functional activity of these chemokines remains to be determined. A dendogram illustrating the relative genetic divergence of the human chemokines is shown in Figure 2; murine chemokines is shown in Figure 3. A dendogram comparing both species with the virally derived chemokines is shown on Figure 4.

The C chemokine: lymphotactin We will first discuss the C subfamily of chemokines since it has only one member. Although the C subgroup just has one cysteine bond, it is functional. Lymphotactin is the name for the only characterized C chemokine (Kelner et al., 1994). Alignment of the amino acid sequence shows that this chemokine is missing one of the two N-terminal cysteines as well as the corresponding third cysteine. Although it does not have all the conserved cysteines, it does have chemotactic activity, indicating that the conserved amino acid substitutions in lymphotactin are able to maintain the functional conformation (Table 1). The lymphotactin receptor has recently been characterized and named XCR1 (Yoshida et al., 1998).

988 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl

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 initiation site. Alignment performed by Dr Robert Stephens of the Frederick Biomedical Super Computer Group SAIC, Frederick, NCI-FCRDC.

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

Figure 2 Dendogram of human chemokines.

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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  subfamilies Chemokine

Chromosome location

Cell sources

Lymphotactin (C) (LPTN)

1

CD8 > CD4 T cells, mast cells, NK1.1 CD4+ T cells

Fractalkine (CX3C) (neurotactin)

16q13

Stimulants

Major in vitro effects

Major in vivo effects

Attracts thymocytes, DC, T and NK cells

Lymphocyte trafficking

Augments antitumor effects

EC

TNF

Microglial cells

IL-1

Transmigration of endothelial cells by mononuclear cells Attracts T, M, N, and NK cells

M

Brain inflammation Adhesion to endothelial cells Nerve repair

Acts on microglia and astrocytes IL-8

4

M

LPS

N

Mitogens

F

Particulates

EC

Viruses

K

Bacteria

NK

IL-1

T

TNF

SMC

IL-3

Ep

IL-13

Activate neutrophil, chemotaxis, adhesion, shape changes, degranulation, enzyme release, and respiratory burst Endothelial cell proliferation Inhibits IL-4-mediated IgE production Inhibits interferon antiviral effects

Mobilize BM neutrophils Antibacterial host defense Acute and chronic inflammation Angiogenesis Modulates hematopoiesis Promotes viral replication

H2O2 IL-7 Hypoxia UVB GRO(, , )/ MGSA

4

M

LPS

Neutrophil activation

Acute inflammation

F

IL-1

Melanoma cell proliferation

Fibroplasia

EC

TNF

Angiogenesis Endothelial cell proliferation

Mesothelial cells

Fibroblast proliferation Activates T cells NAP-2

4

Plt

Platelet

M

activation

Neutrophil activation

Acute inflammation Clot resorption

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

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Table 1 (Continued ) Chemokine

Chromosome location

Cell sources

Stimulants

Major in vitro effects

Major in vivo effects

ENA-78

4

K

LPS

Endothelial cell proliferation

Acute inflammation

F

IL-1

Angiogenesis Neutrophil activation

M

Mobilize BM neutrophils

TNF

EC SMC GCP-2

4

Osteosarcoma cells

Neutrophil gelatinase- release

Acute inflammation

EC, endothelial cells; M, monocytes; N, neutrophils; F, fibroblasts; Plt, platelets; SMC, smooth muscle cells; K, keratinocytes; Ep, epithelial cells; T, T cells; NK, natural killer cells; DC, dendritic cells.

it is membrane-anchored (Bazan et al., 1997; Pan et al., 1997). Many of the other chemokines merely tether noncovalently to glycosaminoglycans (GAG), as will be discussed. In addition to the chemokinex domain at the N-terminus, fractalkine contains a mucin stalk, linked to transmembrane and cytoplasmic domains. Although fractalkine is expressed as a tethered cytokine, a shed, soluble 95 kDa form has also been detected. The soluble 76 amino acid recombinant chemokine component of fractalkine is functional and can activate pertussis toxin (PTX)-sensitive Ca2‡ flux and induce chemotactic responses by cells expressing the monospecific fractalkine receptor, CX3CR1 (Table 1 and Table 2). Neurotactin, which was independent identified in 1998, is a murine homolog of fractalkine, and is highly expressed in both the central nervous system and in peripheral neurons, whereas astrocytes and microglial cells express CX3CR1 (Harrison et al., 1998). Tethering by the mucin domain appears indirectly to facilitate activation of the chemokine domain (Imai et al., 1997). Fractalkine also directly induces adhesion of leukocytes to endothelial cells in a PTX-resistant and -specific manner. Consequently, the adhesion mediated by cell-associated fractalkine appears to occur independent of activation of the chemokine domain.

STRUCTURE±FUNCTION RELATIONSHIPS OF CHEMOKINE LIGANDS A prominent secondary structural feature of chemokines is the triple-stranded antiparallel sheet that

forms a sheet floor for the hydrophobic C-terminal  helix to lay across. The N-terminus up to the first cysteine does not have an ordered structure in solution, with the exception of RANTES which has a small region of strand just before the first cysteine (Skelton et al., 1995). The spacial relationship of the N-terminus, sheet and  helix is maintained by two disulfide bonds. The three structural domains have distinct functions. The N-terminus is essential for activation of G protein-coupled receptors (Gong and Clark-Lewis, 1995). The N-terminus and the sheet provide both a scaffold for quaternary interactions resulting in high-affinity ligand binding to G proteincoupled receptors and the formation of chemokine dimers. The C-terminal  helix of some chemokines has been shown to interact with low-affinity (Kd ˆ 10ÿ5 M) GAGs which are found on cell surfaces and matrix proteins (Witt and Lander, 1994). The basic charge of these ligands accounts for their heparin-binding properties. Although not essential for function, the GAG-binding capabilities of the chemokines are reported to facilitate receptor interactions and the haptotactic migration of receptor-bearing cells over matrix proteins and cellular faces. A number of chemokines contain consensus sites for N-linked and/or O-linked glycosylation. The naturally produced chemokines are heavily glycosylated but the bacterially produced recombinant proteins, which lack glycosylation, retain receptor-binding activity and induce calcium flux and chemotaxis both in vitro and in vivo. The in vivo function of glycosylation remains to be determined, but it may play a role in prolonging the chemokine half-life, and may improve GAG binding.

Table 2 Leukocytes targeted by chemokine±receptor interactions Chemokine ligands

Functional chemokine receptors

Neutrophils

Resting T cells

IL-8 > GCP-2  NAP-2

CXCR1

X

X

IL-8, GRO, ENA-78, GCP-2, NAP-2

CXCR2

X

X

X

X

IP-10, MIG, I-TAC, (SLC+)

CXCR3

SDF-1, SDF-1

CXCR4

BLC (BLR-1 ligand)

CXCR5 X

Activated T cells

T cell subsets

NK cells

X

TH1  TH2

X

X X

RO+, IL-2R

X

CD8

B cells

Monocytes

X

X

X

X

Dendritic cells

CX3CR1

MIP-1, RANTES, HCC-1, HCC-2, MCP-2, MCP-3, MCP-4, MPIF1

CCR1

X

MCP-1, MCP-2, MCP-3, MCP-4

CCR2

X

Eotaxin 1, eotaxin 2, RANTES, HCC-2, MCP-2, MCP-3, MCP-4, MIP-1

CCR3

X

TH2, IL-4 producers

X

TARC, MDC

CCR4

X

TH2, RO+

X

MIP-1 , MIP-1, RANTES, MCP-2

CCR5

X

TH1 > TH2

X

LARC (MIP-3)

CCR6

X

X Mature

X

X

X

X

Immature

X

X

SLC, ELC (MIP-3 )

CCR7

X

X

X

Immature

X

Immature

X

TH1, CD4, CD8

X

Mature

TH2

CCR8

X

XCR1

X

X X

X

X

X X

CD4

I-309, TARC, MIP-1

Eosinophils

X

Fractalkine (neurotactin)

Lymphotactin

Basophils

Immature

X

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors Figure 5 Ribbon structure of human IL-8 overlain with RANTES. The similar regions of IL-8 (amino acids 12±26/41±53) and RANTES (amino acids 14±28/41±53) are shown in blue. The N-terminal and C-terminal regions of IL-8 are shown in yellow. The N-terminal and C- terminal regions of RANTES are shown in magenta. The data was taken from PDB (Abola et al., 1989) database numbers 1RTO (Skelton et al., 1995) and 3IL8 (Rajarathnam et al., 1994). Graphic was generated by Dr Nagarajan Pattabiraman of the Frederick Biomedical Super Computer Group SAIC, Frederick, NCI-FCRDC using Insight 95, Molecular Simulation Inc. (Full colour figure can be viewed online.)

The solution and/or crystal structures of a few chemokines have been determined. Several generalizations can be made concerning the chemokine tertiary structure. The chemokine monomer conformation is globular. The spacing of the first two cysteines results in a difference in the orientation of the loop formed between the N-terminus and the first strand. In the CXC chemokines, this loop has a right-hand hook compared to the left-hand spiral found in CC chemokines. This results in an increased spacing between the N-terminus and the sheet in the CC chemokines (Abola et al., 1989), as shown in Figure 5. Differences in electrostatic potential and hydrophobicity differences have been used to explain the different quaternary structures observed for CC and CXC chemokines (Covell et al., 1994). The chemokines form dimers at micromolar concentrations, under conditions of low pH and high salt concentration. Two modes of dimerization have been detected and these were called the IL-8 and MIP-1 dimer forms because these were two of the first chemokines for which solution structures were determined. It appears that the CC chemokines typically

993

exhibit the MIP-1 dimer form and the CXC chemokines produce the IL-8 dimer form. However, the CC chemokines MCP-1 and MCP-3 can form both IL-8 and MIP-1 -like dimers (Lubkowski et al., 1997; Meunier et al., 1997). In addition, the crystal structure of stromal derived factor (SDF-1) reveals a monomer with both CXC and CC structural properties, supporting the notion that SDF-1 is a unique primordial chemokine (Dealwis et al., 1998). Solution studies of PF4 and a MIP-1 mutant show that these chemokines from stable tetrameres (Mayo et al., 1995; Dealwis et al., 1998; Laurence et al., 1998). In conclusion, all the chemokine monomers are very similar, with the exception of the N-terminal loop, 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 chemotaxis (Lusti-Narasimhan et al., 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, but are no longer chemotactic for monocytes (Beall et al., 1992). These data demonstrate a close association between primary structure and function. Chemokines have well-defined structural and functional domains. Interaction between the chemokine ligand and the chemokine receptor occurs at several sites. A high-affinity binding interaction occurs between the N-terminus, before the first turn, of the chemokine and the N-terminus (the so-called first extracellular domain) of the chemokine receptor (Samson et al., 1997). The activation of the receptor is stimulated by amino acids located in the turns (Clark-Lewis et al., 1994). One structural feature of the receptors that is essential for proper folding is the disulfide bond between extracellular domains. The residues involved in the activation site on the receptor appear to vary among the chemokines. For example, the residues of CCR5 that are essential for activation by RANTES do not appear to be identical to those used by MIP-1 (Samson et al., 1997). The sites determining specificity appear to be located in extracellular domains 1 and 2 of the receptor. However, sites of activation appear to reside in domains 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 from the activation sites on the extracellular domains of the chemokine receptors.

Biologically functional ligand unit The identity of the biologically functional form of the chemokines is quite controversial. Bioassay observations indicate that chemokines can act either as monomers or as multimers in vivo. The functional chemokine form is likely to be the multimer as it binds to GAG (Stringer and Gallagher, 1997; Spillmann et al., 1998). Both PF4 and IL-8 have been shown to bind heparin sulfate; however, the only structural model meeting the charge and position constraints of this interaction involves chemokine multimerization. It appears that GAG binding requires the dimer form of IL-8 and the tetramer form of PF4. The PF4 concentration at sites of trauma has been determined to be 170 mM, indicating that local concentrations of PF4 could favor tetramer formation and association with GAG molecules (Stringer and Gallagher, 1997). This issue is complicated even further by the possibility that the chemokine receptors may dimerize. Several lines of evidence favor the monomer as the functional form of the chemokine. Structural analysis conditions require very high concentrations of protein - levels 10- to 1000-fold greater than the protein concentrations needed for biological function, which may not occur in vivo. Also, mutational changes can be made in the primary structure that prevent multimerization and do not significantly affect function (Rajarathnam et al., 1994). Finally, the primary structure of vMIP-II, a virally encoded (human herpesvirus 8 or HHV8) chemokine, resembles that of MIP-1; however, vMIP-II fails to dimerize regardless of the pH or protein concentration (personal communication from Barry L. Schweitzer).

activating peptide 2 (NAP-2). PBP is not chemotactic, but progressive proteolysis produces TG and NAP2, which are chemotactic (Walz et al., 1989). CTAPIII has been reported to antagonize NAP-2 function. Thus, N-terminal proteolysis provides a level of regulation of chemokine function. The mature secreted chemokine may be further cleaved extracellularly by a number of enzymes. For example, CD26 is a dipeptidylpeptidase, which recognizes the GP site expressed on the surface of activated leukocytes. Recent work has shown that this peptidase removes the first two amino acids from the N-terminus of RANTES and GCP-2 (Proost et al., 1998a). Truncated RANTES is inactive in chemotaxis assays and has a greatly attenuated ability to induce calcium flux. However, truncated RANTES maintains a high affinity for RANTES receptors. Thus, the N-terminally deleted RANTES acts as a natural antagonist for RANTES. Similar results have been observed with SDF-1; however, the CD26-truncated form of GCP-2 has been found to retain activity. Nterminal truncated versions of MCP-1, MCP-2, and MCP-3 have been isolated from mononuclear cells (Proost et al., 1998b). The MCP-2 deletion, missing the five N-terminal residues, is a functional inhibitor of chemotaxis. The peptidase that truncates the MCPs remains to be identified.

PATHOPHYSIOLOGICAL AND DEVELOPMENTAL ROLES OF CHEMOKINES Rather than discussing each chemokine individually we will discuss them in groups based on their structural relationships and major functional activities.

Posttranslational regulation

Role of CXC (ELR‡) chemokines in acute inflammation

Chemokines are produced as propeptides and are cleaved to yield the active secreted form. N-terminal proteolysis is necessary to produce the active mature protein and further N-terminal proteolysis inactivates mature chemokines. The peptidases that clip the proform, yielding the functionally mature form, have not been fully characterized. Perhaps one of the most complicated posttranslational modifications is the sequential processing of platelet basic protein (PBP) to produce connective tissue-activating protein (CTAPIII), -thromboglobin ( TG), and neutrophil

Some of the CXC chemokines, including IL-8, NAP-2, ENA-78, GRO, GRO , GRO , and GCP-2, contain a tripeptide motif at N-terminal positions 4, 5, and 6 composed of glutamic acid, leucine, and arginine (Table 1). This ELR motif is essential for highaffinity binding to CXCR2 (Schraufstatter et al., 1993; Clark-Lewis et al., 1994) (Table 2). Only IL-8, and to a lesser extent GCP-2, bind CXCR1 with high affinity. These ELR‡ CXC chemokines are produced by a wide variety of cells in response to many stimulants, most notably proinflammatory cytokines such

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

995

Table 3 Phenotypes of murine chemoattractant receptor knockouts Targeted gene

Phenotypic abnormalities

IL-8R homolog (CXCR1 and CXCR2)

Defective neutrophil chemotactic response to MIP-2 and KC. Increased bone marrow myelopoiesis with splenomegaly, lymphadenopathy, and plasmacytopoiesis only in conventional germ-laden environment. Elevation of serum IgE. Reduced resistance to bacterial challenge

CCR1

Defective granulomatous response to Schistosoma mansoni eggs. Decreased neutrophil antifungal response with lower TNF levels. Reduced mobilization of progenitor cells from the bone marrow. Protection from pancreatitis-induced pneumonitis

CCR2

Severely reduced monocyte recruitment in peritoneal inflammation model. Reduced TH1 responses. Increased susceptibility to Listeria monocytogenes. Decreased atherogenesis

CCR5

Mice are endotoxin-resistant and have greater CMI response. Mice have reduced resistance to challenge with L. monocytogenes. Humans with defect have less of rheumatoid arthritis and are resistant to HIV infection

CXCR4

Similar to SDF-1 knockout, but also noted defective mesenteric angiogenesis and impaired cerebellar development

CXCR5

Lack inguinal lymph nodes, have small Peyer's patches, abnormal primary follicles, and lack germinal centers. B cells are diffusely distributed in T cell areas. Impaired humoral immune responses

C5a

Mobilizes reverse passive Arthus reactions in lung. Partially suppresses neutrophil infiltration, edema formation, TNF, and IL-6 production in reverse passive Arthus reactions of peritoneal cavity and skin

PAF receptor

Reduced anaphylactic response to antigens. Intact endotoxin shock response

as IL-1 and TNF. Since CXCR1 and CXCR2 are expressed on neutrophils, it accounts for the capacity of these chemokines to induce acute inflammatory responses and to be highly expressed at sites of acute inflammation. The ELR‡ CXC chemokines promote the adherence of neutrophils to endothelial cells (EC), their subsequent diapedesis, and migration along a gradient of chemokines associated with matrix proteins and cell surfaces toward inflammatory sites (Taub et al., 1996). As would be expected, mice with targeted disruption of the IL-8 receptor homolog have reduced acute inflammatory responses to irritants, e.g. carrageenan (Table 3) (Cacalano et al., 1994). These IL-8 receptor-deficient mice also exhibit reduced resistance to challenge by some microbial pathogens. At higher chemokine concentrations, polymorphonuclear leukocytes are activated to produce oxygen radicals and to degranulate (Taub et al., 1996). Several granule proteins, including the defensins, azurocidin, and cathepsin G, are themselves chemotactic for mononuclear cells and presumably serve as signals that help convert acute into chronic inflammatory responses and thus promote the conversion of innate into adaptive immune responses (Chertov et al., 1996, 1997). Thus, chemokines have the potential to activate a number of downstream

effector molecules with the capacity to amplify further inflammatory and immunological host defenses. Neutrophil degranulation may be achieved more readily by IL-8-induced activation of CXCR1 than CXCR2 (Jones et al., 1997). Consequently, the release of these neutrophil granule-derived effector molecules may be a unique function of IL-8.

Angiogenic effects of CXC chemokines The ELR‡ CXC chemokines are also angiogenic and promote in vivo neovascularization in studies employing rabbit cornea and in vitro in chicken chorioallantoic membranes (Keane et al., 1998). Moreover, tumors that constitutively secrete IL-8, GRO, or ENA-78, when exposed to neutralizing antibodies to these chemokines, show reduced growth and metastatic spread based on interference with neovascularization (Keane et al., 1998). It is noteworthy that IFN, IFN , and IFN , which are known to have angiostatic effects, all suppress the production of ELR‡ CXC angiogenic chemokines, but stimulate the production of ELRÿ CXC chemokines, such as IP-10 and MIG, which are angiostatic. Consequently the angiostatic effects of interferons may be mediated by

996 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl Table 4 Properties of human CXC (ELRÿ) chemokine subfamily Chemokine

Chromosome Cell sources location

Stimulants

Major in vitro effects

Major in vivo effects

PF4

4

Plt aggregation

Inhibits endothelial cell proliferation

Angiostatic

Plt

Procoagulant Attracts fibroblasts IP-10

4

M TF EC

Immunostimulant IFN, IFN , IFN , Inhibits EC TNF, LPS Angiostatic Attracts activated T cells, NK cells, and Antitumor immunity monocytes

K Mesothelial MIG

4

M

IFN

Inhibits EC Attracts activated T cells

Hepatic cells I-TAC

4

Astrocytes

Angiostatic Host resistance to bacterial infection

IFN , IL-1 (astrocytes)

Attracts T cells

Constitutive

Attracts naive more than memory T cells

M T SDF-1

10

BM stroma, widely expressed

Competes with T tropic HIV-1

Attracts CD34+, EC, Platelet development, B cell development, progenitor cells, and myelopoiesis B cells, and megakaryocytes Angiogenesis and lymphocyte trafficking BLC/BCA-1 4

Spleen, lymph node, Constitutive liver, appendix

this shift from the production of angiogenic to angiostatic CXC chemokines. Furthermore, avian species have also been shown to produce angiogenic chemokines. The chicken 9E3/ CEF4 gene product, which is similar to human IL-8, is however chemotactic for monocytes and T cells and is also angiogenic (Martins-Green and Hanafusa, 1997). The mechanistic basis for the modulation of angiogenesis by chemokines remains somewhat controversial. The ELR‡ CXC chemokines are reported to be chemotactic for endothelial cells derived from smaller blood vessels that are reported to express CXCR1 (Schonbeck et al., 1995) and for endothelial cells from sites of wound healing that express CXCR2 (Nanney et al., 1995). However, endothelial cells from human umbilical veins and larger blood vessels such

Attracts B cells and subset of CD45RO+, IL-2RA+ T cells

B cell homing

as aortas do not express these receptors and the angiogenic effects of these chemokines on HUVECs has been proposed to be indirect (Petzelbauer et al., 1995). Since there is no deficiency in vascularization in IL-8 receptor knockout mice, the angiogenic effects of ELR‡ CXC chemokines appear to be entirely redundant. Although most of the ELRÿ CXC chemokines have been found to be angiostatic, SDF-1 and SDF1 are notable exceptions and have recently been found to be potent angiogenic factors (Tachibana et al., 1998) (Table 4). These CXC chemokines are alternative splice variants of the SDF gene and are similar in activities. SDF-1 is mitogenic and chemotactic for all types of endothelial cells, and expression of CXCR4 by endothelial cells is enhanced by the

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors classical angiogenic factors VEGF and bFGF (Salcedo et al., 1999). Human SDF-1 promotes the sprouting (outgrowths) of rat aortic endothelial cells in vitro and induces small blood vessel formation at subcutaneous sites of injection in mice (Salcedo et al., 1999). SDF-1 is primarily essential for the development of mesenteric vasculature since it is defective in CXCR4 knockout mice (Table 3), presumably because there are no redundant compensatory angiogenic mediators available at that locale (Tachibana et al., 1998).

Angiostatic chemokines Most of the ELRÿ CXC chemokines, including PF4, IP-10, MIG, and I-TAC, are angiostatic. Although PF4 was the first chemokine to be described, it is the only chemokine that is not chemotactic for leukocytes (Oppenheim et al., 1991). PF4 has weak chemotactic effects only on fibroblasts, is a weak downregulator of hematopoiesis (Daly et al., 1995), and has some procoagulant activity. PF4 is present in platelet granules and, like NAP-2, is released by inducers of platelet aggregation. It appears that PF4 is antiangiostatic (Gengrinovitch et al., 1995), and on that basis has been clinically evaluated for its antitumor effects in humans (regrettably, unsuccessfully to date) (Tanaka et al., 1997). Based on indirect evidence it has been suggested that PF4 suppresses certain B cell functions (Crisi et al., 1996). The endogenous receptor for PF4 has not been identified, but PF4 does bind to the HHV-8 encoded chemokine receptor (Murphy, 1997). The other members of the ELRÿ CXC chemokine subfamily, IP-10, MIG, and I-TAC, all use CXCR3, are chemotactic for activated T cells and monocytes, and both IP-10 and MIG have potent angiostatic effects (Cole et al., 1998; Keane et al., 1998). IP-10 and MIG appear to act in a nonredundant manner in vivo. MIG is expressed largely by the liver, primarily in response to IFN , and is systemically distributed (Farber, 1990), whereas IP-10 is expressed more widely and is believed to be active at local sites of inflammation. For example, overexpression of IP10 is observed in several inflammatory dermatological disorders, namely contact dermatitis and eczema. I-TAC is also widely expressed constitutively in the thymus, spleen, placenta, lung, liver, and pancreas. IP-10 has been shown to have antitumor activity based both on interference with angiogenesis and induction of T cell-dependent antitumor immunity with subsequent resistance to rechallenge by the same tumor (Luster and Leder, 1993). The antitumor effects reported for IL-12 may be mediated through

997

the production of IFN and the subsequent induction of angiostatic IP-10 and MIG (Tannenbaum et al., 1998). The antiangiogenic effects of IP-10 may also account for the capacity of this chemokine to impair wound healing (Luster et al., 1998).

Homeostatic and developmental CXC chemokines The CXC chemokine known as SDF-1 was initially identified as a pre-B cell growth-stimulating factor (PBSF). SDF-1 behaves more like a homeostatic hematopoietic hormone that is critical for homing and angiogenesis than like a proinflammatory chemokine (Nagasawa et al., 1996a). SDF-1 is considered a more primitive chemokine since it is located on chromosome 10, separate from all the other CXC chemokines which are located on chromosome 4, and therefore probably did not arise by gene duplication. Furthermore, SDF-1 exhibits equidistant sequence homology to CXC and CC chemokines and, unlike other chemokines which are highly divergent, SDF-1 is highly evolutionarily conserved with only a one amino acid difference between humans and mouse (Nagasawa et al., 1996b). Low levels of SDF-1 are constitutively present in normal plasma (Bleul et al., 1996). SDF-1 is expressed by a wide variety of cells and tissues and is chemotactic for a broad spectrum of target cells because the receptor (CXCR4) is widely distributed on neutrophils, monocytes, T and B cells, CD34‡ hematopoietic progenitor cells, bone marrow-derived dendritic cells, megakaryocytes, endothelial cells, neurons, astroglial cells, and gastrointestinal epithelial lining cells (Nagasawa et al., 1996b; Aiuti et al., 1997). SDF-1 is essential for life since deletion of CXCR4 (Table 3) or SDF-1 (Table 5) by homologous recombination in mice yields pups that die soon after birth (Tachibana et al., 1998; Zou et al., 1998). These mice exhibit defective B cell development, abnormalbonemarrowmyelopoiesis,ventricularseptal defects, defective gastric vascularization (Tachibana et al., 1998), and impaired cerebellar development (Zou et al., 1998). BLC or BCA-1, the only known ligand for CXCR5 (BLR-1) has unique noninflammatory functions and, as inferred from CXCR5 knockout mice, is primarily involved in B lymphocyte homing and the development of normal lymphoid tissue organization (Gunn et al., 1998a; Legler et al., 1998). BLC is constitutively expressed at a high level in the follicles of Peyer's patches, liver, spleen, and lymph nodes. BLC induces Ca2‡ flux and is chemotactic for B cells and for a small subset of memory (CD45RO‡ IL-2R‡) T cells.

998 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl

Table 5 defects

Phenotypes of mice with chemoattractant gene

Targeted gene

Phenotypic abnormalities

SDF-1

Mice die before or shortly after birth with immunodeficiency due to lack of B lymphocytes, impaired myelopoiesis, and cardiac ventricular septal defect. Mesenteric blood vessels are defective

MIP-1

Reduced pulmonary mononuclear infiltrate to challenge with influenza virus resulting in higher titers of influenza virus Less severe autoimmune myocarditis after coxsackie viral infection Knockout has increased MODS mortality and CTL activity Reduced NK-mediated inflammation

Eotaxin

Diminished number of circulating eosinophils and 60% reduction in tissue eosinophils in response to an allergen challenge in lung and cornea

MCP-1

Defective in vivo monocyte recruitment Reduced resistance to bacterial challenge (e.g. Listeria monocytogenes)

C5

Defective granulocyte inflammatory responses Impaired mast cell degranulation Deficient inflammatory TNF response

5-Lipoxygenase

Resistant to PAF-induced lethal shock Maintain sensitivity to endotoxin lethality Blocked production of leukotrienes

CXCR5 was actually identified prior to the ligand and by knockout technology CXCR5 was revealed to have surprising functions (Forster et al., 1996) (Table 3). Mice with homologous deletion of the BLR1 (CXCR5) gene lacked inguinal lymph nodes, and had diminished Peyer's patches, abnormal primary lymphoid follicles and lacked germinal centers (GC). Activated B cells failed to home to follicles and were instead retained in the T cell areas of the lymphoid tissues. Despite the presence of B cells, these mice were found to be hyporesponsive to antigenic stimulation. These data provided the first evidence that some chemokines are essential in development for microenvironmental homing of immune cells. BLC is therefore also representative of a class of homeostatic regulatory cytokines rather than being a proinflammatory CXC chemokine.

Subgroup of proinflammatory and allergenic CC chemokines The CC chemokines in general target mononuclear cells rather than neutrophils and are either proinflammatory (Table 6) or homeostatic mediators (Table 7). The members of the MCP/eotaxin subgroup of CC chemokines are closely related in chromosome location, gene and protein structure and receptor utilization (Berkhout et al., 1997). This subgroup is of particular interest to allergists because these CC chemokines include some potent eosinophil attractants and a number with histamine-releasing capabilities (Luster and Rothenberg, 1997). MCP-1 was initially discovered as an IL-1-induced product of mononuclear cells with potent monocyte chemotactic activity (Sauder et al., 1984, Yoshimura et al., 1987). MCP-1 shares 65% of the amino acid sequences of MCP-2, MCP-3, and MCP-4 and, while all of these chemokines utilize CCR2, MCP-2, MCP-3, and MCP-4 also use CCR1 and CCR3, and MCP-2 also binds to CCR5 (Table 2). They all attract basophils and mast cells and can rapidly induce basophil degranulation with histamine release (Luster and Rothenberg, 1997). Eotaxin 1 utilizes solely CCR3 and, like MCP-4, is a potent attractant of eosinophils and basophils. Although the above outline of the cellular targets of this chemokine subgroup provides considerable basis for their contribution to allergic reactions, this needs to be put in perspective by pointing out that, like most chemokines, these allergenic CC chemokines are pleiotropic in their functions. They have other proinflammatory activities: MCP-1, MCP-2, MCP-3, and MCP-4 are monocyte and T cell attractants that participate in chronic inflammatory reactions. Deletion of CCR2 results in defective monocyte chemotaxis and impaired host defense against infectious challenges (Kurihara et al., 1997, Kuziel et al., 1997). Deletion of the MCP-1 gene has similar consequences (Lu et al., 1998) (Table 5). In contrast, transgenic mice engineered to overexpress MCP-1 in their keratinocytes become hypersensitive to cutaneous irritants and develop increased delayed-type hypersensitivity reactions to contact sensitizing agents (Nakamura et al., 1995). Although MCP-1 attracts macrophages to tumors, it only induces transient partial tumor regression. By contrast, MCP-3transfected tumor cells attract a leukocyte infiltrate that includes some dendritic cells and leads to the development of tumor immunity (Fioretti et al., 1998). A more recently discovered member of this subgroup of CC chemokines, known as MPIF2 or

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

999

Table 6 Properties of human proinflammatory CC chemokine subfamily Human CC chemokines, alias

Chromosomal Tissue and location cell sources

Exogenous and endogenous stimulants

Major effects

MCP-1/MCAF

17

Mo, astrocytes, mesothelial, EC, Os, microglia, airway SMC, F, Ep, K, PBMC, Mc, Eo

IFN , IL-1, TNF, IL-6, IL-13, SCF, IL-15, H. pylori, bacteria, respiratory syncytial virus, other viruses

Activates Mac, BS and MC

Small intestine, heart, PBMC, astrocytes, lung, skeletal muscle, pancreas, thymus, mesenchymal, F

IL-1 , IFN

Activates Mac, prevents HIV infection

MCP-2

17

Histamine release Favors TH2 polarization

Releases histamine Attracts BS and MC

MCP-3

17

Mo, EC, PBMC, astrocytes IL-4, IL-13, mycobacteria

Activates Mac, attracts BS and MC Releases histamine Induces tumor immunity

MCP-4

17

Mac, small intestine, colon, EC

Releases histamine and LTC Attracts eosinophils, BS, MC, M

RANTES

17

Eo, Ep, mesothelial, F, SMC, Mc, K, T cells, Mo, EC, platelets

IL-1 , TNF, angiotensin II, respiratory syncytial virus, HIV, influenza

Activates T, increases integrin adhesion, prevents HIV infection Attracts Eo and BS, histamine release

MIP-1/ LD78, stem cell inhibitor

17

Mo, microglia, fibroblasts, Lc, Mc, T cells

Activates T, increases LPS, IL-1 , TNF, IL-2, IL-6, respiratory integrin adhesion, syncytial virus inhibits BM progenitor proliferation, prevents HIV infection Augments TH1 polarization

MIP-1 /Act-2, HC21 17

Microglia, fibroblasts, Mc, T cells, Mo

LPS, IL-1 , TNF

Activates T, increases integrin adhesion, prevents HIV infection

PARC/DC-CK-1, MIP-4, AMAC-1

17

Lung, lymphoid organs, Mo-derived-DC, Mac

LPS IL-4, IL-13, IL-10

Attracts naõÈ ve CD45 RA‡ T cells and EOS

Eotaxin

17

EC, Mo, Ep, T, Mac, F

IL-4, TNF

Recruitment of Eo in skin diseases, strongly correlated with allergic and chronic nasal inflammation Modulates hematopoiesis

Eotaxin 2/MPIF2

7

Activated T, Mac

GM-CSF, anti-CD3/IL-2

Eo filtration in vivo, induces histamine and LTC4 from BS, inhibits BM progenitor proliferation

I-309

17

Mac, Ms, T

IgG, LPS

Tumor immunity, promotes TH2 responses Antiapoptotic for thymoma cells

Table 6 (Continued ) Human CC chemokines, alias

Chromosomal location

Tissue and cell sources

Exogenous and endogenous stimulants

Major effects

MPIF1/CK 8 CK 8-1 splice variant

17

Aortic EC, lung, liver, BM and placenta, Mac

IFN and IFN

Inhibits BM progenitor proliferation Attracts activated T cells and Mo

HCC-1/ splice variant HCC-3

17

Wide distribution/Lymphoid tissues, muscles, BM

HCC-2/ Lkn1, MIP-5

17

Mo, liver, small intestine, colon, fetal liver

Constitutive

BM progenitor proliferation Attracts T cells, Mo and EOS Inhibits BM progenitor proliferation

LEC/HCC-4, ILINCK, LMC

17

Liver, Mo

IL-10

Attracts T cells and Mo Inhibits BM progenitor proliferation

EC, endothelial cells; Mo, monocytes; MC, mast cells; N, neutrophils; F, fibroblasts; Plt, platelets; SMC, smooth muscle cells; K, keratinocytes; Ep, epithelial cells; T, T cells; NK, natural killer cells; DC, dendritic cells; Eo, eosinophils; Os, osteosarcoma; BS, basophils; BM, bone marrow; Mac, macrophages; Lc, Langerhans cells.

Table 7 Properties of human homeostatic and developmental CC chemokine subfamily Human CC chemokines, alias

Chromosomal location

Tissue and cell sources

MDC/STCP-1

16q13

Monocyte-derived-DC, thymus, lymph node, appendix, mature macrophages

Exogenous and endogenous stimulants

Major effects

Attracts myeloid DC and IL-2 activated NK cells May favor TH2 responses

LARC/MIP-3, Exodus

2

Lymphoid tissue, liver, thymus, EC, EOS, appendix, tonsil, PBL, monocytes and DC

Constitutive

Inhibits BM progenitor proliferation Induces memory T adhesion, attracts DC

ELC/MIP-3

9

Thymus, lymph node, tonsil, appendix, anti-IL-10-treated monocytes

Constitutive

Induces naõÈ ve T cell adhesion and homing to parafollicular areas

SLC/6Ckine, Exodus-2, TCA-4

9

Lymph node, appendix, spleen, small intestine, thyroid, thymus, DC, monocytes

Constitutive Augmented by IL-10

T cell and DC homing to lymphoid tissue

TARC

16q13

Thymus, lung, colon, and small intestine, PBMC

PHA

May favor TH2 responses

TECK

Not mapped in human on mouse 8

Thymus, DC, thymic progenitors

LPS

T development in fetal thymus Attracts Mo, splenic DC, thymocytes

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 1001 eotaxin 2, clearly functions as an allergenic chemokine because it uses CCR3 (Forssmann et al., 1997). Eotaxin 2 is located by itself on chromosome 7 and may be a more primitive member of the MCP group that did not arise by gene duplication. Eotaxins 1 and 2 exhibit 39% amino acid sequence homology. Eotaxin 2 is chemotactic for basophils and eosinophils and induces histamine release from IL-3-primed basophils. Although eotaxin 2 is somewhat less potent than eotaxin 1, they completely cross-desensitize each other and MCP-4. Eotaxin 2 has no chemotactic effects on other leukocytes. Although I-309 is located near the subgroup of allergenic chemokines on the dendogram, it is not usually considered an allergenic cytokine. Nevertheless, it may participate in promoting IgE-dependent responses since I-309 uses CCR8 (Roos et al., 1997; Tiffany et al., 1997) which is preferentially expressed on CD4‡ TH2 cytokine-producing cells (Zingoni et al., 1998). I-309 also induces chemotaxis of monocytes, NK cells, and immature dendritic and B cells. Transfection of tumor cells with TCA3, the murine I-309 homolog, increases tumor cell immunogenicity and leads to tumor rejection. I-309 is a unique chemokine in having antiapoptotic activity that protects BW 5147 thymoma cells from dexamethasone-induced lysis (Van Snick et al., 1996). This activity has not been confirmed for normal thymocytes or T cells. A second subgroup of homologous proinflammatory CC chemokines includes RANTES, MIP-1, MIP-1 , and PARC (Dc-Ck1). RANTES, like MCP1, MCP-2, MCP-3, and MCP-4, also attracts basophils and induces histamine release (Table 6). RANTES, and at times MIP-1, attract eosinophils, but they are not considered major contributors to allergic responses, since neutralizing antibodies to RANTES and MIP-1 do not reduce allergic responses significantly (Luster and Rothenberg, 1997). RANTES preferentially attracts CD45RO‡ memory T cells and has more potent chemotactic effects on IL2-activated T cells since these cells express more functional chemokine receptors. Thus, RANTES is a participant in chronic immunologically regulated inflammatory responses. Higher micromolar concentrations of RANTES have been reported to be mitogenic for T lymphocytes, and to induce lymphokine production and the expression of IL-2R (Bacon et al., 1995). This T cell response is herbimycin A- rather than pertussis toxinsensitive, suggesting the involvement of non-Gi coupled STM receptor-dependent signal transduction by tyrosine kinases (Bacon et al., 1995). The physiological relevance of this observation remains to be established.

MIP-1 is a more potent attractant of mononuclear cells than MIP-1 , perhaps because it interacts with CCR1, CCR3, and CCR5, while MIP-1 acts primarily through CCR5. Despite the enormous redundancy of proinflammatory chemokine and cytokine mediators, MIP-1 knockout mice exhibit diminished postcoxsackie virus autoimmune myocarditis and show defective host defenses against influenza virus challenge (Cook et al., 1995). The pulmonary mononuclear inflammatory infiltrates are diminished in these mice, but their influenza virus titers are greatly increased (Table 5). Related studies show that deletion of the CCR1 gene increases the susceptibility of mice to Aspergillus infection and decreases their granulomatory inflammatory response to challenge with eggs of Schistosoma mansoni parasites (Gao et al., 1997). Furthermore, mice with a CCR1 gene defect exhibit elevated resistance to acute respiratory distress syndrome (ARDS) associated with acute pancreatitis (Table 3). This is accompanied by lower levels of TNF, and is consistent with the proinflammatory role of CCR1 and the CCR1 ligands (Gerard et al., 1997). Although PARC is related at the primary sequence level to the other proinflammatory chemokines, the functional relationship of PARC to proinflammatory chemokines is still unclear and the receptor for this chemokine remains unknown. PARC is expressed in the lung and lymphoid organs and produced by dendritic cells located in germinal centers and T cellrich areas of secondary lymphoid organs (Gosse et al., 1997). Other investigators have detected expression of PARC by alveolar and other macrophages (Hieshima et al., 1997a). PARC is unique in that it is induced by TH2 cytokines such as IL-4, IL-13, and IL-10, but not by IFN . PARC is preferentially chemotactic for naõÈ ve CD45RA‡ T cells and may therefore participate in early steps of immune induction. The HCC subgroup of CC chemokines also exhibits proinflammatory functions. HCC-1 actually has 46% homology with MIP-1 and MIP-1 , but it is constitutively expressed in normal spleen, liver, gastrointestinal tract, skeletal and heart muscle, and bone marrow and is present at 1±10 nM concentrations in plasma (Schulz-Knappe et al., 1996). Although HCC-1 utilizes CCR1, it is a weak activator of calcium flux only for monocytes and induces CD34‡ myeloid progenitor cell proliferation (Table 6). In contrast, HCC-2 (or leukotactin 1) expression is restricted to the gut and liver (Pardigol et al., 1998). It is a CC chemokine with six cysteines and a putative third disulfide bond. HCC-2 utilizes CCR1 and is functionally similar to MIP-1 with potent chemotactic effects on monocytes and

1002 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl lymphocytes and modest effects on eosinophils. HCC2 is unusual in that it is also chemotactic for neutrophils. HCC-3 is a splice variant of HCC-1, and remains to be functionally characterized. HCC-4 is unusual in that its expression by monocytes is upregulated byIL-10, which downregulates most other chemokines and cytokines (Hedrick et al., 1998). HCC-4 is chemotactic for PBMCs, the THP1 monocytic cell line and T lymphocytes. However, the receptor(s) for HCC-4 remains to be identified. Another member of this subgroup, MPIF1, is also most similar in sequence to MIP-1 (Patel et al., 1997). MPIF1 is prominently expressed by lung, liver, bone marrow, and placenta, and is chemotactic for IL-2-stimulated (not anti-CD3 activated) T cells and monocytes. The gene that encodes MPIF1 yields an alternative splice variant, producing a second functional chemokine known as CK 8-1 (Youn et al., 1998). Both splice variants are potent agonists of CCR1 and CK 8-1 is chemotactic for monocytes, lymphocytes, and neutrophils.

Developmental and homeostatic CC chemokines Several CC chemokines have recently been identified that may play more important roles in development, homeostatic trafficking, and homing of various lymphoid subsets rather than in inflammation (Table 7). They include TECK, MDC, TARC, SLC (6C-kine), ELC (MIP-3 ), and LARC (MIP-3). TECK is uniquely located by itself on mouse chromosome 8. TECK is a CC chemokine which is highly expressed in the adult thymus, developing fetal spleen, small intestine, and fetal medullary thymus by lymphoid-derived dendritic cells with MHC class II and CD11‡ expression. On the other hand, TECK is not expressed by bone marrow-derived dendritic cells (Vicari et al., 1997). The receptor for TECK is unknown. However, TECK is chemotactic for monocytes, splenic DC, and thymocytes. These observation suggest that TECK may be more involved in lymphoid trafficking to and in the thymus rather than in proinflammatory responses. It has been proposed that TECK may induce the migration of thymocytes from the thymic cortex to the medulla and may be important in the repopulation of thymus following irradiation. MDC is located on chromosome 16 along with TARC, with which it shares 37% amino acid identity and utilization of CCR4 (Table 2). MDC is unusual in that it is a late product of macrophage lineage cells that have been cultured up to a week. MDC is not

expressed by monocytes, but only by macrophages and dendritic cells. It is most highly expressed in the thymus, and to lesser extent in spleen, lung, and small intestine (Godiska et al., 1997). In contrast with the proinflammatory chemokines, MDC is not induced by TNF, PHA, or PMA. Even though MDC and TARC are the only known ligands for CCR4, they only partially overlap in their functions (Table 7). MDC desensitizes Ca2‡ flux by TARC on CCR4transfected cells, but not the reverse. MDC is chemotactic for monocyte-derived dendritic cells and for IL-2-activated NK cells. Monocyte responses require 100-fold higher concentrations of MDC than are needed to attract dendritic cells. TARC is also highly expressed in the thymus. Since neither the genes for TARC or MDC have undergone targeted disruption, the physiological role of these chemokines remains rather speculative. The homeostatic chemokines (SLC, ELC, and LARC) are unusual in that they are constitutively produced by lymphoid tissues and are located on chromosome 9, or 2 in the case of LARC, rather than chromosome 17 along with the other proinflammatory CC chemokine genes (Rossi et al., 1997). These chemokines direct the homing of T cells and mature dendritic cells to their sites of localization in lymphoid tissues. SLC is also called 6C-kine because it has two additional C- terminal cysteines (Hedrick and Zlotnik, 1997). Dendritic cells and anti-IL-10-treated monocytes express SLC, but lymphocytes do not. SLC appears to be a pivotal director of T cell homing (Gunn et al., 1998b). SLC selectively interacts with CCR7 on thymocytes, naõÈ ve, and memory CD4 and CD8 T cells and mature dendritic cells. SLC is expressed on the surface of high endothelial venules (HEVs) and on endothelial cells of lymphatics in liver and small intestine where it rapidly (within seconds) induces adhesion of naõÈ ve T cells to ICAM-1, and promotes the transmigration of recirculating T lymphocytes into secondary lymphoid tissue (Campbell et al., 1998b; Gunn et al., 1998b). Subsequently, in conjunction with another CCR7 ligand, ELC (MIP3 ), SLC induces T cells to migrate into the T cell-rich perifollicular areas (Yoshie et al., 1997, Campbell et al., 1998a). This conclusion is based in part on studies revealing that an experiment of nature, the plt mouse that is deficient in lymph nodes and T cells, could not be restored to an immunocompetent state by adoptive transfer of normal T cells (Bacon and Oppenheim, 1998). Yet, T cells from such plt mice are not defective and maintain the immunocompetence of wild-type mice, suggesting a stromal defect in plt mice. In fact, plt stromal cells completely fail to express SLC and were partially deficient in ELC due to a genetic defect at a site on mouse chromosome 4

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 1003 closely linked to the SLC locus. T cells fail to migrate to the white pulp of the spleen as a consequence of these defects, while the content of dendritic cells in white pulp is also reduced and they cluster outside the white pulp. Since activated mature dendritic cells express CCR7, their migration from the periphery into lymphoid tissues is impaired. Consequently, SLC and ELC are important for directing resting T cells and activated dendritic cells to appropriate sites in lymphoid tissues. In addition to being chemotactic, SLC may also suppress hematopoiesis (Hromas et al., 1997). SLC may also have angiostatic activities, since it is the first CC chemokine reported to have lowaffinity interactions with a CXC receptor (CXCR3) (Soto et al., 1998). The gene for homeostatic chemokine LARC (MIP3), is located by itself on chromosome 2. LARC is the exclusive ligand for CCR6 (Greaves et al., 1997; Power et al., 1997). It is produced peripherally by tissues such as the liver (Hieshima et al., 1997b) and inflamed skin and is found in the outer mucosal surface of lymph nodes. LARC is also widely expressed by lymphoid tissues. LARC is preferentially chemotactic for CCR6-expressing CD4‡ resting T cells and CD1a‡ CD86- immature peripheral dendritic cells such as Langerhans cells (Power et al., 1997). Dendritic cells derived from CD34‡ myeloid progenitor cells, and monocyte-derived dendritic cells (De Yang et al., unpublished observations), exhibit a chemotactic response to LARC, while neutrophils and monocytes do not. When dendritic cells are activated by stimulants such as LPS, TNF, IL-1, and IFN , their CCR6 expression is downregulated, while CCR7 expression is upregulated. Consequently, as dendritic cells mature they become unresponsive to LARC, but become responsive to lymphoid tissuederived SLC and ELC (MIP-3 ) and migrate into lymphoid tissue to initiate T cell-dependent immune responses (Sozzani et al., 1997, Dieu et al., 1998).

Role of chemokines in adaptive immune responses Besides the crucial consequences of regulating homing of T and B cells and dendritic cells, chemokines also influence adoptive immune responses based on their activating effects on APCs and enhancement of T helper cell functions. A number of the CC chemokines, including MIP-1, MIP-1 , MCP-1, MCP-3, and RANTES, have been shown to act as costimulators of T cells. This is evident from their capacity to augment anti-CD3-stimulated T cell expression of IL2R, IL-2 production, and proliferation (Taub et al.,

1996). These data are reinforced by observations that anti-RANTES antibodies partially reduce polyclonal responses of T cells, suggesting that induction of endogenous RANTES is a normal participant in T cell activation. These immunological effects of chemokines may be attributable in part to an increase in the expression of costimulatory molecules such as B7 on APCs. Furthermore, antigen-presenting cells such as dendritic cells are a rich source of chemokines that act on T cells. In addition to being a source of proinflammatory chemokines such as RANTES, MIP-1, MIP-1 , and MCP-1 to MCP-4, myeloid dendritic cells are an important source of TARC, MDC, and PARC (Dc-Ck1). TECK is produced by lymphoid dendritic cells. A number of chemokines such as MCP-1, MCP-3, MIP-1, MIP-1 , and RANTES promote CTL- and NK cell-mediated killing of target cells, presumably by augmenting the process of killer cell degranulation with release of serine esterase and enhanced 2-integrin-dependent binding of the killer to the target cell (Taub et al., 1995). The augmentation of killer cell activation is blocked by pertussis toxin and is deficient in MIP-1 knockout mice (Cook et al., 1995). MIP-1, MCP-1, and other chemokines also contribute to polarization of TH1 and TH2 immune responses, respectively (Karpus and Kennedy, 1997). MCP-1 favors the conversion of TH0 cells to TH2 cytokine-producing cells in vitro (Karpus and Kennedy, 1997). In vivo administration of MCP-1 favors the production of IL-4 while reducing IL-12 levels and may thus indirectly exacerbate IgEdependent allergic humoral immunity (Karpus and Kennedy, 1997). In contrast, homologous deletion of CCR2 unexpectedly reduces TH1-type cytokine responses (Boring et al., 1997; Gao et al., 1997). MIP-1 has immunoregulatory activity and is capable of polarizing TH0 and TH1 cytokine-producing cells, while suppressing IL-4 and promoting IFN -dependent cell-mediated immune responses (Karpus and Kennedy, 1997). Additional definitive data concerning the preferential effects of chemokines have been generated by showing the selective expression of chemokine receptors on T cell subsets. Thus, TH1 cells preferentially express CXCR3, CCR5, and CCR7, while TH2 cells preferentially express CCR3, CCR4, and CCR8 (Bonecchi et al., 1998; Sallusto et al., 1998; Zingoni et al., 1998). Consequently, TARC, which binds to CCR4 and CCR8, and MDC, which shares CCR4 (both receptors which are preferentially expressed on the TH2 cells), may preferentially engage in immune deviation to humoral immunity. There are some reservations concerning these observations in that CCR4 is said to be expressed on the pro-TH2 as well as mature TH2 cells, while CCR3 is

1004 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl expressed exclusively on the minority of IL-4-producing TH2 cells and also a few TH1 polarized cell lines. Nevertheless, these observations are consistent with data showing that ligands restricted to utilizing these receptors favor either cellular or humoral immune responses. Thus, IP-10 and MIG, which are induced by IFN and use CXCR3, are prominent participants in TH1-type cellular-mediated immune reactions. In contrast, eotaxin and MCP-4, which utilize CCR3, promote TH2-type humoral immunity. The knockout mice with defective receptors for IL-8 homologs exhibit marked elevation of IL-4 levels, suggesting that IL-8-like signals normally suppress this TH2 cytokine.

Hematopoietic effects of chemokines Many of the chemokines may also participate in the regulation of hematopoiesis. A number of the chemokines have been shown to have direct effects on GM-CSF- and SCF-induced hematopoietic colonyforming assays. IP-10, PF4, MIP-1, MCP-1, eotaxin 2, LARC, murine MRP-1 and MRP-2, and MPIF1 have all been reported to suppress hematopoietic colony formation (Broxmeyer et al., 1995; Youn et al., 1995; Patel et al., 1997), while SDF-1, eotaxin, HCC-1, murine MIP-2, and IL-8 can enhance early hematopoietic progenitor cell growth (Keller et al., 1994; Nagasawa et al., 1996a). Eotaxin can be detected early in murine development in the embryonic yolk sac and CCR3 first appears in development on embryonic hematopoietic cells (Quakenbush et al., 1998). Eotaxin acts in synergy with stem cell factor to increase the number of fetal hematopoietic colonies in vitro, but under other conditions eotaxin may actually inhibit hematopoietic progenitor cell growth (Quakenbush et al., 1998). On the other hand, eotaxin 2 is also an inhibitor of hematopoietic progenitor cell colony formation. The single IL-8 receptor homolog of both CXCR1 and CXCR2 in mice has been deleted by homologous recombination (Table 3). Neutrophils from these deficient mice no longer show chemotactic responses to human IL-8 or to the functional mouse homologs of IL-8 known as KC and MIP-2 (Cacalano et al., 1994). These knockout mice, if housed in a conventional animal environment, develop splenomegaly, bone marrow myeloid hyperplasia, and lymph node hyperplasia with an increase in neutrophils, B cells, and plasma cells. These phenotypic abnormalities fail to develop in a germ-free environment (Shuster et al., 1995). Presumably, microorganisms present in the

normal environment stimulate a compensatory myeloid and B cell hyperplasia in these IL-8 receptordeficient mice, suggesting that the ELR‡ murine chemokines normally downregulate signals leading to myeloid and B cell generation (Czuprynski et al., 1998). The suppressive effect on myeloid progenitor proliferation is synergistically accentuated by chimeric mutants of IL-8 and PF4, and this supports a role for these chemokines in the regulation of hematopoiesis (Daly et al., 1995). Except in the case of SDF-1 which is documented to be required for the development of normal bone marrow myelopoiesis, the in vivo relevance of MIP1, MCP-1, CCR1, CCR2b, CCR3, or CCR5 remains dubious, because mice with homologous deletion of these genes do not exhibit defective hematopoietic development. However, these observations may be due to the existence of redundant compensatory mediators. In fact, CCR1 knockout mice following endotoxin challenge do have decreased myeloid progenitor cells in the spleen and circulation, possibly due to impaired mobilization from the bone marrow (Gao et al., 1997). Despite these caveats, investigators are attempting to exploit members of the HCC subgroup of chemokines that are involved in regulating hematopoiesis as therapeutics. For example, HCC-4, like MIP-1, suppresses myeloid progenitor cell proliferation. Furthermore, both MPIF1 and its splice variant CK 8-1 suppress hematopoietic cell colony formation. Although clinical trials with MIP-1 have been disappointing, MPIF1 is also being evaluated in animal models for its capacity to counteract bone marrow suppressive chemotherapy (Patel et al., 1997; Bacon and Oppenheim, 1998). Pretreatment with MPIF1 is protective and reduces the lethal hematopoietic toxicity of chemotherapy, presumably by preventing bone marrow precursor cell cycling. The eventual utility of this approach and whether this is the best chemokine for this application remain to be established.

BIDIRECTIONAL INTERACTIONS OF CHEMOKINES AND MICROBIAL ORGANISMS It is a paradigm that a major role of proinflammatory chemokines is to mobilize innate and adaptive immune responses against infectious agents. However, some observations are difficult to reconcile with this paradigm. For example, interferons suppress the production of IL-8, and IL-8 in turn suppresses the antiviral activity of IFN in association with a reduction

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 1005 in 20 50 -A oligo-adenylate synthetase activity (Khabar et al., 1997). Paradoxically, IL-8 promotes replication of viruses such as cytomegalovirus (CMV) by IL-8, which is of dubious benefit for the host. Furthermore, microorganisms have subverted some of the chemokine receptor defense mechanisms to their own advantage (Horuk and Peiper, 1996; Murphy, 1996, 1997). For example, Plasmodium vivax parasites utilize the DARC receptor (known as the Duffy antigen) as their site of invasion of human red blood cells (Chitnis et al., 1996). HIV-1/HIV-2 retroviruses require certain chemokine coreceptors along with CD4 as sites for invasion of human leukocytes and microglia (Littman, 1998). Monocytotropic (NSI) HIV-1 strains utilize CCR5 as a coreceptor for the invasion of monocytes and dendritic cells (Table 8). RANTES, MIP-1, MIP-1 , and MCP-2, by virtue of their interaction with CCR5 can competitively block cellular invasion by monocytotropic strains of HIV-1 (Cocchi et al., 1995; Gong et al., 1998). A minority of HIV isolates can utilize CCR2, CCR3, CCR8, or even a CMV-encoded analog of the CCR5 coreceptor encoded by the US28 gene (Pleskoff et al., 1997). However, although as many as 13 chemokine receptors and related STM receptors can function as HIV coreceptors in transfected cells, only three (CCR5, CCR8, and CXCR4) have proved to have this capacity in primary cells. Only one, namely, CCR5, has actually been shown to affect pathogenesis of HIV. Humans homozygous for mutated nonfunctional forms of CCR5 (e.g. CCR5
32) are almost always resistant to infection with HIV-1, while heterozygotes have a longer latency period before developing AIDS. Population genetic studies have also shown that CCR5
32 heterozygotes with rheumatoid arthritis are subject to less inflammation and less severe symptomatology (Garred et al., 1998). However, CCR5 gene deletion makes mice more susceptible to bacterial challenge, more endotoxin resistant and they develop greater cell-mediated immune (CMI) and humoral immune responses to antigenic challenge (Zhou et al., 1998). Consequently, in view of the relatively mild effects of deletion of the CCR5 gene (unlike CXCR4), CCR5 is viewed as an attractive target for the development of anti-HIV agents that may be free of major detrimental side-effects. Both CXCR4 and CCR5 can mediate T cell, thymocyte, microglial, and dendritic cell infection by HIV. CXCR4 (fusin), the receptor for SDF-1, is used by lymphocytotropic (X-4) strains of HIV-1 as a coreceptor for T cell and neuronal invasion (Amara et al., 1997). Consequently, SDF-1 can competitively interfere with the invasion and subsequent intralymphocytic replication of HIV-1 in T cells. Poly-

morphism of a single base (SDF1-30 A) in the 30 UTR of the SDF-1 RNA is reported to have a protective effect on disease progression of some cohorts of HIV1‡ seroconverters (Winkler et al., 1998). The basis for this observation remains unclear. A number of pox and herpesviruses also produce homologs of chemokine receptors and chemokines, which function either as analogs (Table 8) or inhibitors (Table 9), presumably to the virus's advantage. The chemokine-like products largely cluster between the CXC and CC subfamilies or between lymphotactin and the HCC-1-HCC-4 subgroup (Figure 4). The myxoma members of the poxvirus family have been shown to produce several gene products that bind chemokines and other cytokines (Lalani and McFadden, 1997). The secreted myxoma M-T7 gene product, although homologous to the binding domain of the IFN R, ubiquitously interacts with the heparin-binding domains C-terminal  helix of CXC and CC chemokines (Table 9). Deletion of the M-T7 gene results in a nonpathogenic virus which induces greater leukocyte infiltration with diminished viral replication, resulting in the survival of the rabbit host. The M-T1 myxoma gene product more selectively inhibits the activation of monocytes by CC chemokines. Deletion of this M-T1 gene only modestly increases host resistance and increases monocytic infiltration of the virally injected sites, but does not reduce viral replication markedly. The basis for the inhibitory effect of M-T1 remains unclear, but it does bind CC chemokines with higher affinity than the CXC chemokines. The molluscum contagiosum members of the poxvirus (MCV) family characteristically induce noninflamed papular skin tumors in children and AIDS patients (Table 8). One of the MCV products acts as a broad-spectrum antagonist that blocks the response of a variety of leukocytes to both CXC and CC chemokines which may account for the failure of host inflammatory responses to MCV (Damon et al., 1998). This gene product also suppresses replication of hematopoietic progenitor cells (Krathwohl et al., 1997). Murine CMV code for a gene product with homology to chemokines, which is predicted to promote the evasion of host defense by CMV (MacDonald et al., 1997). Human herpes CMV genes encode 1, a CXC-like chemokine with 35% homology to IL-8 which induces Ca2‡ flux and chemotaxis of monocytes (Schall, unpublished). The Nterminus has an ELR sequence at positions 2, 3, and 4. The 1 ligand competitively inhibits ELR chemokine interactions with CXCR2, but not CXCR1 or the CCR5-like CMV-derived US28 receptor-like gene product.

1006 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl

Table 8 Virokines: viral gene products mimicking chemokines Virus (virus class)

Protein name

Similar to human chemokine(s)

Target or function

HHV-8 ( herpesvirus), also called KSHV or KSVH

vMIPI

MIP-1 (38.0% identity)

Blocks HIV-1 entry into human mononuclear cells Strongly angiogenic in CAM. Infects Kaposi's sarcomas

HHV-8 or KSVH

vMIPII

MIP-1 (45.1% identity)

Chemoattractant for CCR3-expressing cells Blocks HIV-1 entry through CCR3, CCR5, and CXCR4 Strongly angiogenic in CAM. Induces calcium flux in CCR1, CCR2, CCR3, CCR5, and CXCR4-expressing cells

HHV8

vMIPIII (BCK)

Only limited CC chemokine homology

MCVI (orthopoxvirus)

MC148R1

Homologous to CC chemokines including MIP-1

Blocks MIP-1-induced chemotaxis and inhibits hematopoietic progenitor proliferation. Also blocks IL-8, MCP-1, etc.

MCVII

MC148R2

Homologous to CC chemokines including MIP-1

Blocks MIP-1-induced chemotaxis and inhibits hematopoietic progenitor proliferation less well than MCVI

HIV-1 (retrovirus)

Tat

No significant homology

Agonist for CCR2 and CCR3 but not CCR1, CCR4, or CCR5

HIV-1 monotropic (5) (NSI) strains

gp120 gp41

Structural similarity to MIP-1, MIP-1 , and RANTES

Agonist for CCR5, CCR2, CCR8, and US28

HIV-1 lymphotropic (4). Strains

gp120 gp41

Structural similarity to SDF-1

Agonist for CXCR4

CMV

?

CXC chemokine ELR containing

Calcium flux in neutrophils and neutrophil chemotaxis

CMV

?

CXC chemokine Non-ELR containing

Stealth virus ( herpesvirus)

CC chemokine

Sequence similarity; no known function as yet

HHV-6 ( herpesvirus)

CC chemokine

Sequence similarity; no known function as yet

CAM, chorioallantoic assay.

HHV-8 has been isolated from Kaposi's sarcomas of AIDS patients. This Kaposi's sarcoma-associated herpes virus (KSHV) produces chemokine-like gene products called vMIP-I and vMIP-II (Table 8).

vMIP-I, like its MIP-1 homolog, may inhibit X5 strains of HIV by competing for CCR5 (Moore et al., 1996). vMIP-II has 40% homology to human CC chemokines and binds CC chemokine receptors, but it

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 1007 Table 9 Virally encoded inhibitors of chemokine Type of inhibitor

Origin

Function

Viral chemokine inhibitor VCCI/ORFB

Orthopoxvirus

High-affinity inhibitory binding of all chemokines

Cowpox virus p32 G3R

Orthopoxvirus

High-affinity inhibitory binding of all chemokines and low-affinity binding of IL-8

Variola virus (human smallpox virus) p35 C23L

Orthopoxvirus Vaccinia virus

High-affinity inhibitory binding of eotaxin, RANTES, MCP-1, and MIP-1 but not IL-8, GRO, or ENA-78

p35 RPV

Leporipoxvirus (rabbit pox)

Removal of vCCI gene results in increased mononuclear cell infiltration into infected tissues and reduced virus burden

S-T1

Shope fibroma

Removal of vCCI gene results in increased mononuclear cell infiltration into infected tissues and reduced virus burden

M-T7

Poxviruses

Resembles IFN receptor but binds to and inhibits CXC, CC, and C chemokines with low affinity

Myxoma virus S-T7

Shope fibroma

Resembles IFN receptor but binds to and inhibits CXC, CC, and C chemokines with low affinity

M-T1

Poxviruses

Binds CC chemokines

Myxoma virus

also has considerable binding affinity for CXCR4 and competitively inhibits binding of SDF-1 (Kledal et al., 1997). vMIP-II also blocks the calcium flux and chemotactic effects of both the RANTES interaction with CCR5 and MCP-3 with CCR3. vMIP-II therefore behaves as an HHV-8-derived antagonist. However, vMIP-II is actually a partial agonist since it induces signal transduction in eosinophils via CCR3 and CCR8 (Boshoff et al., 1997). More recently, vMIP-I and vMIP-II, unlike MIP-1 and MIP-1 , were found to be angiogenic (Boshoff et al., 1997) and this presumably serves to promote Kaposi's sarcoma tumor growth (Ganem, 1997). KSHV also encodes BCK, a third CC chemokine homolog, which inhibits apoptosis of thymoma cells (Blo5147), presumably by interaction with CCR8, in a manner similar to I-309.

INHIBITORS OF CHEMOKINE ACTIVITIES Obviously, the possibility that viruses and other agents may provide useful chemokine inhibitors is under intensive study (Table 10). Endogenous cytokines and hormones such as IL-10, TGF , and glucocorticoids all suppress the production and effects of proinflammatory chemokines (Howard et al., 1996). Investigation of the effects of chemokine gene deletions and neutralizing antichemokine antibodies have revealed that interference with chemokines can result in dramatic antiinflammatory effects despite the redundancy in chemokine ligands (Baggiolini and Moser, 1997). For example, neutralizing anti-IL-8

1008 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl

Table 10

Targets and effects of chemokine inhibitors

Inhibitor name

Target

Effect

IL-10

Regulates chemokine mRNA expression

Reduces proinflammatory chemokine expression such as IL-8 and MCP-1. May stimulate regulatory chemokines like HCC-4

TGF

Regulates chemokine mRNA expression

Reduces proinflammatory chemokine expression in most cell types

Glucocorticoid

Regulates chemokine mRNA expression and protein production

Reduces RANTES expression by nasal fibroblast, epithelial cells, and keratinocytes, IL-8 expression in epidermis and eosinophils, MCP-1 expression by keratinocytes and eosinophils

Anti-IL-8

Blocks IL-8 binding to CXCR1 and CXCR2

Protects against glomerulonephritis

Anti-MCP-1

Blocks MCP-1 binding to CCR2

Reduces inflammation associated with arthritis

Opiates

Regulates chemokine receptor-transduced signal

Reduces chemokine-induced migration in vitro

PAF antagonists (several small molecules and modified PAF variants)

Block PAF binding to specific receptor and PAFR signal

Reduce inflammation

RANTES mutants and AOP-RANTES

Blocks ligand binding to CCR1, CCR3, and CCR5

AOP-RANTES inhibits HIV coreceptor activity of CCR5, mutant RANTES block eotaxin function in vivo

SB-225002

Blocks IL-8 binding to CXCR2

Blocks IL-8-induced neutrophil migration in vivo

2-2 dipheny-5-(4-chlorophenyl) piperidinyl valeronitrite

Blocks CCR1 ligands

Useful to block chronic inflammation

NSC 651016 Ureido derivative of distamycin

Negative agonist for CXCR4, CCR1, CCR3, CCR5, and CCR8

Blocks chemokine-induced calcium flux in vitro.

ALX40 nonapeptide of D-arginine

Blocks CXCR4

Blocks SDF-1-induced calcium flux and T-tropic HIV infection in vitro

AMD3100 a bicyclam

Blocks CXCR4

Blocks SDF-1-induced calcium flux and T-tropic HIV infection in vitro

T22 octadecapeptide derived from horseshoe crab

Blocks CXCR4

Blocks SDF-1-induced calcium flux and T-tropic HIV infection in vitro

antibodies ameliorate hypoxia-induced reperfusion injuries following vascular occlusion and can reduce the inflammation that causes ARDS (Harada et al., 1994). Anti-MIP-1 reduces chronic relapses in EAE, while antimurine MIP-2 can block the initiation of acute EAE, but can also increase susceptibility to bacterial infections in mice, as reviewed in Oppenheim et al. (1996). Many mutated variants of chemokines have been generated that are partial or complete antagonists (Baggiolini et al., 1997). To date, aminooxypentane-RANTES has been determined to be a potent antagonist for RANTES and is being evaluated for its therapeutic effects in animal

Blocks HIV-1 infection in vivo. Reduces chemokine-induced inflammation in vivo

models of a variety of autoimmune diseases (Mack et al., 1998). In vivo enzymatic cleavage may result in posttranslationally modified chemokines which can either be more potent or behave as antagonists of chemokines (Proost et al., 1998a). A number of low molecular weight pharmacological agents have been shown to interfere with chemokine receptor interactions. They include an antagonist of CXCR2 that inhibits IL-8 (White et al., 1998), bicyclams (Schols et al., 1997), and T22 from horseshoe crabs (Murakami et al., 1997), both of which interfere with CXCR4, and a distamycin derivative which inhibits ligands for CXCR4, CCR1, CCR3, CCR5, and CCR8

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 1009 (Howard et al., 1998). Some chemokines such as PARC may actually be partial agonists and may interfere with homologous chemokines such as eotaxin. In addition, there is the phenomenon of desensitization which is probably responsible for the observation that intravenously administered IL-8 actually has an inhibitory effect on inflammatory responses to peripherally administered IL-8 as well as C5a and LTB4 (Gimbrone et al., 1989). An analogous deactivation of monocytes has been reported to occur in mice transfected to overproduce MCP-1 (Rutledge et al., 1995). Macrophages from such mice are hyporesponsive to MCP1, resulting in lower resistance to infectious challenges. This reflects the capacity of chemokines, through heterologous desensitization, to phosphorylate and inactivate other STM. Other agents with immunosuppressive effects include opioids, which also use STM and induce heterologous desensitization (Grimm et al., 1998), as do the gp120 and gp41 envelope proteins of HIV-1 (Ueda et al., 1998; Wang et al., in press). These envelope proteins activate CD4 on monocytes and T cells which in turn activates kinases that phosphorylate and interfere with the chemotactic responses of these cells by internalizing a number of STM coreceptors for chemokines as well as for fMLP. Overall, considerable effort is aimed at identifying molecular means of regulating chemokine-mediated reactions and many of them will be discussed in greater detail in the overview chapter on chemokine receptors and in the chapters devoted to individual chemokines.

OTHER PROINFLAMMATORY CHEMOTACTIC FACTORS Although other chemoattractant factors have temporarily taken a back seat to the intense interest in chemokines, they play unique and important roles in host defense. Obviously, small tripeptides such as fMLP derived from bacterial cell walls can potentially serve as an early warning signal that attract phagocytic cells to sites of infection. Comparisons of the attractive potency of fMLP, C5a, and proinflammatory chemokines reveal the former to be more potent and to deviate phagocyte migration away from chemokines (Campbell et al., 1996). It should be noted that a number of formylated peptides like fMLF are chemotactic, but fMLP has been studied most intensively as a prototype (Schiffmann et al., 1978). Two receptors, one with high and the other with lower affinity for fMLP, have recently been cloned.

Activation of the complement cascade yields the C5a and C3a anaphylatoxins. Studies indicate that C5a is more potent and a more important chemoattractant than C3a (Gallin et al., 1992). In fact, experiments of nature have generated C5a-deficient mice that can still produce C3a, but exhibit defective neutrophil accumulation at sites of inflammation (Table 5). Although C5a can presumably be generated only after an immune response has led to antibodyantigen interaction induction of the classical complement pathway, C5a can also be generated rapidly by alternate pathways in response to stimuli such as LPS. Consequently, C5a can contribute to the rapid mobilization of phagocytic cells early in host defense. C5a has a diversity of proinflammatory activities in addition to leukocyte chemotaxis, including the promotion neutrophil adhesion to endothelial cells, degranulation of phagocytes, and induction of cytokines such as IL-1, IL-6, IL-8, and TNF. Decomplementation and C5 deficiency block mast cell degranulation and TNF release in response to an inflammatory signal. C5a interacts with a widely expressed C5a anaphylatoxin receptor, a member of the G protein-coupled STM. Targeted disruption of this receptor gene totally prevents lung injury, but only partially decreases the neutrophil infiltration, edema formation, TNF and IL-6 production in reverse passive arthus reactions of the peritoneal cavity and skin (Table 3) (Hopken et al., 1997). Activation of inflammatory leukocytes also releases nonhistamine-containing slow-reacting substance of anaphylaxis (SRS-A) which induced a slow contractile response of nonvascular smooth muscle. Over the past two decades a group of fatty acid arachidonic acid metabolites, known as cysteinyl leukotrienes, have been identified as the molecules responsible for SRA-A activities. A series of molecules are generated in response to activation of leukocytes by proinflammatory cytokines by the 5-lipoxygenase pathway, including LTC4 and its metabolites LTD4 and LTE4 (Lam and Austen, 1992). These leukotrienes have vascular permeability effects, stimulate mucus secretion and bronchial smooth muscle constriction, and may therefore be pivotal contributors to airway obstruction in pulmonary inflammatory diseases. Furthermore they induce vascular contraction and generalized plasma leakage as well as reduced renal output. Finally, LTA4 and LTC4 are intermediates that can be metabolized to form biologically active lipoxins. Lipoxin A4 (LXA4) has chemotactic effects on phagocytic cells, may downregulate inflammatory reactions, and is reported to utilize the lowaffinity receptor for fMLP (fMLPR-1), as will be discussed in the chapter on lipoxin A4 receptor (Gronert et al., 1998). Another product of the 5-lipoxygenase

1010 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl pathway is LTB4, a potent proinflammatory leukotriene which induces chemotaxis, aggregation, shape changes and adherence to endothelial cells of granulocytes, eosinophils, and mast cells. LTB4 also activates monocyte chemotaxis, induces cytokine production by T cells, and enhances immunoglobulin production by B cells. LTB4 uses a high-affinity G protein-coupled seven transmembrane receptor (see the chapter on BLTR: the LTB4 receptor). As is true for a number of the CC chemokines, LTB4, lipoxin A4, and lipoxin B4 promote GM-CSF-induced myeloid progenitor cell hematopoietic colony formation (Stenke et al., 1994). However, recent studies of mice with targeted disruption of the 5-lipoxygenase gene failed to reveal any defects in hematopoiesis (Chen et al., 1994). PAF, a chemotactic phosphorocholine derivative, was actually the first phospholipid to be identified as a proinflammatory intercellular signal. PAF is released by inflammatory leukocytes such as mast cells, basophils, macrophages, and neutrophils in response to opsonizing stimuli, fMLP, GM-CSF, and LPS (Ayala and Chaudry, 1996). Endothelial cells produce a cell-associated form of PAF in response to TNF and IL-1. PAF is constitutively produced in the brain and kidney. PAF induces aggregation of platelets and releases histamine and/or serotonin which in turn induces vascular permeability, bronchoconstriction, hypotension, and thrombocytopenia. PAF acts on widely distributed G protein-coupled STM to induce PMN chemotaxis, upregulate adhesion molecules, and augment reactive oxygen production. PAF is chemotactic for eosinophils and basophils and induces PMN degranulation and leukotriene production. PAF also stimulates a cascade of monocyte/ macrophage effects, including the release of oxygen radicals and cell aggregation, primes mononuclear cells for production of proinflammatory cytokines, and induces the production of prostaglandins. Transgenic mice that are engineered to overexpress the PAF receptor are hypersensitive to endotoxin. Despite the fact that injection of PAF mimics many of the anaphylactic symptoms of endotoxin shock, PAF antagonists have benefitted only a subset of patients with gram-negative bacterial infections or marked endotoxemia. Mice sensitized to an antigen with targeted disruption of their PAF receptor gene had a normal phenotype and remained susceptible to endotoxin shock, but showed markedly reduced anaphylactic symptoms in response to antigen challenge (Table 3) (Ishii et al., 1998). Furthermore, mice with targeted disruption of their 5-lipoxygenase gene showed no reduction in the lethality of endotoxin shock, but became resistant to the lethal effects of

PAF-induced shock (Chen et al., 1994). The knockout mice fail to show PAF-induced bronchoconstriction, vasoconstriction, or the release of peptidyl leukotrienes (Table 5). This suggests that leukotrienes are important mediators of PAF-, but not of endotoxininduced shock.

NEUROPEPTIDE MEDIATORS OF IMMUNITY AND INFLAMMATION Neuroendocrine mediation and regulation of immunity and inflammation involves a complex network of cells and soluble factors interacting through adhesion proteins, G protein-coupled receptors (GPCRs) and other specific cell surface proteins. Neural polypeptides are included in this chapter in part because cellular recognition and responses are transduced by GPCRs similar to those for chemokines, complement peptides, eicosanoids, and many other inflammatory mediators. This brief summary will focus on a number of neurally derived peptide and protein mediators for which roles in immunity and inflammation have been supported at functionally relevant concentrations, by expression of specific receptors during responses, and by meaningful alterations in their contributions by receptor-selective agonists and/or antagonists (Table 11). The neural factors with immune and inflammatory activities also influence functions of the vasculature, smooth muscle, secretory epithelial cells and glands, and diverse neural elements contributing to host defense and physiological accommodation.

Neuropeptides and neurotropins Substance P (SP) is an 11 amino acid member of the tachykinin neuropeptide family, which has wellestablished roles in immunity, through actions on T cells, B cells, and macrophages, and is a potent inflammatory mediator, as a result of effects on mast cells, many types of leukocytes, blood vessels, secretory cells, and neurons of the nociceptive system (Brodie and Gelfand, 1992; Goetzl and Sreedharan, 1992; Goetzl et al., 1995; Canning, 1997). Neurokinin A (NKA) is a 10 amino acid peptide of the tachykinin family, with inflammatory and immune effects similar to SP. NKA is more potent than SP as a bronchoconstrictor, but less potent than SP in eliciting microvascular leakage and mucus secretion in lung tissues. This appears to be a function of the relative levels of expression by target cells of the GPCR NK1 for SP and NK2 for NKA. The

Polypeptide mediators of neuroimmunity and neuroinflammation

Mediator

Source

Receptors

MLCT

T cells

B cells

Antibodies

PMNL

Vascular

Secretion

(A) Neuropeptides and neurotropins Substance P (SP)

Sensory neurons

NK1 > NK1

+

+

+

+

+

+

+

Neurokinin A (NKA)

Sensory neurons

NK1 > NK1

+

+

ND

ND

ND

+

+

Corticotropin-releasing factor (CRF)

Hypothalamus

CRF1, CRF21, CRF2

ND

ÿ+

(central effects)- ND ND (peripheral effects)

ÿ+

0

0

Calcitonin gene-related peptide (CGRP)

Sensory neurons

ND

ÿ

+

ND

+ (EOS)

+

+

Vasoactive intestinal peptide (VIP)

Cholinergic neurons

+

ÿ

0

+/ÿ

+

+

+

VPAC1, VPAC2

(B) Factors regulating neuropeptide release or effects Bradykinin

Plasma 2globulins

B1, B2

All +, but indirect and due to release of SP and NKA.

+

+

Serotonin

Neurons, nonhuman basophils

At least 14 5HTR subtypes

ND

+

+

+

+

+

(All secondary to release of SP or CRF)

ND, not determined; +, stimulatory; ÿ, inhibitory; 0, none; MLCT, mononuclear leukocyte chemotaxis.

ND

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 1011

Table 11

1012 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl contrasting relative lack of effect of NKB on inflammation and immunity similarly reflects the low levels or lack of expression of the NK3 GPCR for NKB. SP, NKA, and NKB are derived from preprotachykinins (PPTs) in neuronal cells of ganglia supplying regional nociceptive sensory innervation. Increases in tachykinin levels in ganglia near sites of inflammation are largely attributable to cytokine-evoked upregulation of PPT mRNA (Fischer et al., 1996), but may also reflect appearance of PPT and generation of SP in macrophages infiltrating inflamed tissues (Ho et al., 1997; Killingsworth et al., 1997). As for other neuropeptides, the effective tissue concentrations of SP and NKA are regulated tightly by specific peptidolysis. SP and other tachykinins are degraded principally by a neutral endopeptidase (NEP) (Nadel, 1991). SP, but not NKA, is also cleaved efficiently by angiotensinconverting enzyme. Tissue levels of NEP also are increased substantially by inflammation, both as a result of changes in NEP content of resident cells and influx of NEP-rich leukocytes (Sreedharan et al., 1990). The activities of SP and NKA, which have been reviewed recently, include stimulation of T cell and macrophage chemotaxis, T cell proliferation, cytokine generation by T cells and macrophages, antigen presentation, and production of IgA. NK1 and NK2 GPCRs are very highly expressed in immune cells of Peyer's patches and other gut lymphoid tissues, and thus effects of SP and NKA are much greater in mucosal and other gastrointestinal and respiratory subsets of lymphocytes than in blood lymphocytes (Stanisz et al., 1987; Nakanishi, 1991). The proinflammatory effects of SP and NKA consist of PMN, leukocyte, and eosinophil chemotaxis, mast cell and eosinophil degranulation, vasodilatation and increased microvascular permeability, smooth muscle contraction, especially in respiratory airways, increased mucus secretion, and hyperalgesia. Some effects of SP and NKA are restricted to subsets of inflammatory cells, as for example degranulation of human cutaneous mast cells, but not those in human lungs. NK1 and NK2 GPCRs transduce most of the inflammatory effects, but some are NK receptor-independent, such as mast cell and eosinophil degranulation (Linthorst and Reul, 1998). Abundant evidence implicates SP and/or NKA as critical mediators of some components of asthma, allergic rhinitis, pancreatitis, several types of inflammatory bowel disease, some forms of arthritis, and immune-mediated meningoencephalitis. Corticotropin-Releasing Factor (CRF) CRF is a 41 amino acid hypothalamic stimulant of the biosynthesis and secretion of pituitary

adrenocorticotropic hormone, which leads to increased adrenal glucocorticoid production (Vale et al., 1981) via the so-called hypothalamic-pituitaryadrenal (HPA) axis. CRF secretion may be evoked by TNF, IL-1, and IL-6 effects on the hypothalamus in inflammatory reactions (Chrousos, 1995). CRF is also found in other areas of the central nervous system, lungs, testes, ovaries, intestines, and immune system. CRF may have major roles in behavioral and immune responses to stress, which are independent of pituitary and adrenal involvement (De Souza et al., 1991). Immune alterations attributable to central nervous system-derived CRF include decreased NK cell cytotoxicity, reduced T cell responses to mitogens and antigens, and lowered bone marrow and spleen content of B cells, especially those of the pro-preIgM‡ subset. As a result, both primary (IgM) and recall/memory (IgM/IgG) antibody responses to antigen are suppressed by central CRF. These observations from in vitro and animal studies have been confirmed by studies of CRF-transgenic mice, overexpressing high basal levels of CRF, where NK, T, and B cell activities were modified as predicted by increases in CNS levels of endogenous CRF (StenzelPoore et al., 1995). Central CRF effects on immunity result in part from increased corticosteroids and/or sympathetic neural mechanisms. In contrast, high local tissue levels of CRF promote peripheral immune inflammation in rodent models of arthritis and uveitis, and in human rheumatoid arthritis, thyroiditis and ulcerative colitis (Wilder, 1995). These proinflammatory effects of peripheral tissue CRF were reversed in animal models by anti-CRF-neutralizing antibodies and the CRF1 antagonist antalarmin (Webster et al., 1996). CRF may activate subsets of mast cells in some tissues. Calcitonin Gene-Related Peptide (CGRP) CGRP is stored in large quantities along with substance P in many of the same subsets of afferent nerves and released by some of the stimuli which mobilize SP, but less is known of its pathways of peptidolytic biodegradation. One or more of a subset of GPCRs bind and transduce effects of CGRP, often by coupling to Gs and eliciting increases in [cAMP]i. The most prominent physiological and pathophysiological effect of CGRP is profound and long-lasting vasodilatation, which is a direct consequence of interaction with a high density of smooth muscle receptors (McCormack et al., 1989). In contrast, CGRP has only weak activity for smooth muscle tone and secretion, where receptors are represented only sparsely. The actions of CGRP of leukocytes in inflammation are restricted to eosinophil chemotaxis, which is

Chemotactic Factors, Neuropeptides, and Other Ligands for Seven Transmembrane Receptors 1013 attributable to peptidolytic release of an active fragment, and inhibition ofmacrophage secretory functions (Davies et al., 1992). The identification of CGRP in neurons of the thymus and, at lower levels, of the spleen rekindled interest in its immune effects (Bulloch et al., 1991). Physiological levels of CGRP inhibit proliferative responses of thymocytes and T cells to mitogens, and of T cells to antigens (Wang et al., 1992; Sakuta et al., 1996). The inhibitory effect of CGRP on proliferation of CD4‡ CD8‡ thymocytes was partially due to enhancement of apoptosis. B cells also express CGRP receptors, but less is known of the functional responses of B cells to CGRP. Preliminary data from several studies have suggested that CGRP stimulates B cell differentiation, in part by enhancing the activation of NFB by IL-1. Vasoactive Intestinal Peptide (VIP) VIP is a 28 amino acid neuroendocrine mediator, which is concentrated in cholinergic neurons. As for substance P and CGRP, VIP is present at very high concentrations in the thymus, other immune organs, and lymphoid follicles of the lungs, gastrointestinal tract, and skin (Fink and Weihe, 1988; Ottaway, 1991). Although immunoreactive VIP is found at very low levels in mast cells, basophils, macrophages, and some sets of T cells, much of this VIP-like peptide is not the native 28 amino acid VIP. Full-length VIP is found at functionally relevant nanomolar concentrations in tissue fluids after local antigen challenge in sensitized rodents and in human tissue fluids after respiratory or dermal challenge with antigens of proven reactivity (Wallengren et al., 1987; Kaltreider et al., 1997). T cells and macrophages, but not B cells, recognize and respond toVIP through two highly homologous GPCRs termed VPAC1 and VPAC2, the relative representation of which is controlled developmentally and by exposure to a wide range of cytokines and inflammatory mediators (Goetzl and Sreedharan, 1992). Thymic VIP facilitates survival and differentiation of CD4‡ CD8‡ thymocytes into CD4‡ helper T cells largely through interacting with VPAC2 (Pankhaniya et al., 1997), but little is known of direct effects on development of other immune cells. VIP may enhance involvement of IL-7 in B cell maturation in some system. VIP is a potent chemotactic factor for T cells and macrophages, and supports migration of immune cells across basement membranes and tissues by inducing expression of 1 integrins characteristic of each type of cell and by evoking the production and release of matrix metalloproteinases 2 and 9 (Xia et al., 1996a, 1996b). Each of these effects on T cell movement is

predominantly transduced by VPAC2. In some subsets of T cells, signals from VPAC1 are inhibitory for chemotaxis elicited by chemokines and other factors (Goetzl et al., 1996). Most other effects of VIP on T cells are largely suppressive and thus oppose actions of SP. VIP inhibits T cell generation of IL-2, IL-4, and IL-10, and suppresses T cell-dependent production of IgG and, in some systems IgM (Mathew et al., 1992; Ganea and Sun, 1993; Hassner et al., 1993; Sun and Ganea, 1993; Boirivant et al., 1994). However, VIP consistently increases generation of IgA and IgE, and in some systems augments secretion of IL-5 and IL-6. VIP also enhances generation of IFN by TH1 cells challenged in vitro with antigen, but not with mitogens (Jabrane-Ferrat et al., 2000). The absence of potent VIP antagonists has hindered studies of the roles of VIP in immunity, but recent disruption of VPAC genes in mice may permit better definition of such roles.

Factors regulating neuromediator release or effects Bradykinin (BK) Bradykinin is a nonapeptide generated in plasma from the proteolysis of high and low molecular weight kininogen 2-globulins by plasma and tissue kallikreins, as well as some other immune cell-derived proteases (Bhoola et al., 1992). The B1 and B2 GPCRs for BK are expressed in blood vessels, epithelial cells, smooth muscle, submucosal glands, nerves, and some endocrine cells, but not immune or inflammatory cells. BK is a potent bronchoconstrictor, secretagogue, vasodilator, vascular permeability factor, and activator of nociceptive sensory nerves (Barnes et al., 1998). Although BK may evoke release of thromboxane and other eicosanoids, and of leukocyte chemotactic factors from epithelial and other cells, the principal source of inflammatory and immune activities released by BK is neural, with both cholinergic and peptidergic components. Most inflammatory and immune effects of BK are attributable to release of SP and/or other tachykinins, as evidenced by the suppressive effects of NK-1 receptor antagonists. Since their effects are indirect, separate chapters on bradykinin and serotonin have not been included. Serotonin (5-HT) Serotonergic neurons influence diverse neural, hormonal, and physiological functions of mammals (Saxena, 1995). The inflammatory and immune effects of 5-HT, however, are attributable largely to

1014 Joost J. Oppenheim, O. M. Zack Howard and Edward Goetzl its effects on synthesis, release, and actions of other neuromediators, especially neuropeptides. 5-HT coexists with SP, NKA, and thyrotropin-releasing hormone in spinal autonomic systems, and with SP and CGRP in the CNS (Ward et al., 1994). 5-HT neurons also synaptically contact and regulate peptidergic neurons of the CNS containing VIP, SP, and CGRP. Numerous stimuli evoke release of SP and CGRP from serotonergic neurons, resulting in concentrations of the neuropeptides capable of altering vascular tone and permeability, and initiating regional neuroinflammation (Linthorst and Reul, 1998). Serotonergic neural control of CRF-containing neurons may increase or decrease regional CNS levels of CRF to an extent which alters not only ACTH secretion, but also central control by CRF of diverse immune functions (Reul et al., 1998).

CONCLUSIONS A wide range of neurally derived amines and peptides moderate and regulate immunity and hypersensitivity in diverse tissues. Recognition of these neuromediators and cellular signaling by GFCRs evokes physiological tissue responses, specialized compartmental immune responses, inflammation, and hypersensitivity. Several normal factors lack primary activity, but act potently on immunity and inflammation by releasing neuropeptides at critical sites of reactions. The emergence of useful pharmacological probes and generation of mice with genetic deletions of neuromediation receptors will facilitate elucidation of relevant neuroimmune mechanisms.

ACKNOWLEDGEMENT We are grateful to Dr Joseph A. Hedrick, Schering Plough Corporation, Keniworth, NJ for providing the dendograms; to Dr Philip Murphy, NIAID, NIH, Bethesda, MD for critical review of this manuscript; and to Ms Cheryl Fogle for outstanding secretarial and managerial assistance. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract no. NO1-CO-56000. The contents of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

The publisher or recipient acknowledges right of the US Government to retain a nonexclusive, royaltyfree license in and to any copyright covering the article.

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