Chemokines

Philip M. Murphy

INTRODUCTION

Chemokines constitute a large specialized family of cytokines that regulate immune responses by activating specific G protein-coupled receptors (GPCRs) on leukocytes.¹^,²^,³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹²^,¹³^,¹⁴ Chemokines may act at multiple levels, including immune system development,¹⁵ leukocyte transendothelial migration,¹⁵^,¹⁶^,¹⁷^,¹⁸ leukocyte positioning within microenvironments,¹⁹^,²⁰^,²¹^,²²^,²³ and both phagocyte and lymphocyte activation.²⁴^,²⁵^,²⁶ They may function beneficially, for example in the setting of antimicrobial host defense and tissue repair, or harmfully, for example in the setting of cancer, chronic inflammation, autoimmunity, and infectious disease (Fig. 28.1). Many pathogens produce broad-spectrum chemokine blocking agents, attesting to the importance of chemokines in host defense,²⁷^,²⁸^,²⁹ and some pathogens, most notably human immunodeficiency virus (HIV) and Plasmodium vivax, exploit host chemokine receptors as essential cell entry factors.³⁰^,³¹^,³² Chemokines may also have nonimmunologic functions, including regulation of organ development.³³^,³⁴^,³⁵ Together, these attributes have suggested potential chemokine-based therapeutic opportunities, and there are now two drugs targeting chemokine receptors approved by the U.S. Food and Drug Administration (FDA), the CCR5 antagonist maraviroc (Pfizer) in HIV,³⁶^,³⁷ and the CXCR4 antagonist plerixafor (Sanofi-Aventis) in hematopoietic stem cell mobilization for transplantation in cancer.³⁸

To date,⁴⁸ chemokines and 23 chemokine receptors have been discovered in man. The goal of this chapter is to delineate their molecular properties and provide an overview of current knowledge of how they coordinate innate and adaptive immune responses, focusing on mouse and human. In this regard, it is important to note that the chemokine system may differ substantially among species, even at the genomic level.³⁹^,⁴⁰^,⁴¹ In addition, despite adopting a standard nomenclature in the year 2000,⁴²^,⁴³^,⁴⁴ competing nonstandard aliases continue to be used in the primary literature, confusing even experts in the field. To address these challenges to clear communication, three illustrations are provided. The first defines the chemokine system repertoire at the genomic level in mouse and human, allowing comprehensive and facile identification of orthologues and species-specific elements (Fig. 28.2); the second is a dendrogram depicting the evolutionary relationship of chemokine and chemokine receptor protein sequences in mouse and man (Fig. 28.3); and the third provides the standard nomenclatures approved by the International Union of Immunological Societies for chemokine proteins and by the International Union of Pharmacology for chemokine receptors, linked to nonstandard aliases and protein sequence accession numbers (Tables 28.1 and 28.2). The names assigned by the HUGO Gene Nomenclature Committee are used for the corresponding chemokine system genes (see Fig. 28.2). In most cases, but not all, the approved gene and protein names are the same.

MOLECULAR ORGANIZATION OF THE CHEMOKINE SYSTEM

Chemokine Structure and Classification

The word “chemokine” is a contraction of “ chemotactic cytokine,” which conveys the main function possessed by members of the family. Nevertheless, chemokines are defined by structure, not function, and may have other functions besides inducing cell movement. At the level of primary structure, chemokines occupy a common sector of sequence space bounded by ˜20% identity for any pairwise comparison, and after processing most are 66 to 111 amino acids in length. At the level of tertiary structure, chemokines fold in a highly conserved manner constrained in part by conservatively spaced disulfide-bonded cysteines.⁴⁵ The family is subclassified based on the number and location of these cysteines. All chemokines have at least two cysteines, and all but two have at least four cysteines (Fig. 28.4A). In the four cysteine group, the first two are either adjacent (CC motif) or separated by either one (CXC motif) or three (CX3C motif) amino acids. C chemokines have only two cysteines, corresponding to the location of Cys-2 and Cys-4 in the other groups. Most chemokines are members of either the CC or CXC group; there is only one CX3C chemokine and one (mouse) or two (human) C chemokines. The group motifs are used as roots followed by the letter “L” (for “ligand”) and a number to create standard chemokine names (see Tables 28.1 and 28.2, and Fig. 28.2).⁴⁴ In human names, all letters are capitalized; in the corresponding mouse name, only the first letter is capitalized. Amino acid sequence identity is < 30% between members of the four major chemokine groups, but ranges from ˜30% to 99% among members of the same group, indicating separate evolutionary histories. Although chemokine genes are located on 10 different human chromosomes, most are found in two large clusters, one for CXC chemokine genes on chromosome 4q21 and one for CC chemokine genes on chromosome 17q12 (see Fig. 28.2). These two clusters contain most of the chemokines that mediate inflammatory immune responses, whereas other chemokines, scattered throughout the genome either alone or in
small clusters on chromosomes 1, 2, 5, 7, 9, 10, 16, 17, and 19, mainly subserve homeostatic immune functions. The strongest functional correlate of the four major chemokine groups is receptor specificity: that is, most chemokine receptors bind more than one chemokine but are restricted by group (Table 28.3). With regard to leukocyte specificity, most neutrophil-targeted chemokines are in the CXC group and most of the monocyte-/macrophage-targeted chemokines are in the CC group. Major T and B cell-targeted chemokines can be found in both groups. The leukocyte target specificity of a chemokine may be narrow or broad and is defined by the expression pattern of its cognate receptor(s) (Table 28.4).

FIG. 28.1. Chemokines Coordinate Immune System Development and Immune Responses in Large Part by Inducing Leukocyte Migration Through Activation of G Protein-Coupled Receptors. Other functions induced by chemokines are listed in the green spaces. CK, chemokine.

CC and CXC chemokines can be further subclassified. The ELR subgroup of CXC chemokines is defined by the amino acid motif glu-leu-arg N-terminal to Cys-1 and is encoded by a cluster of genes on human chromosome 4q21 (see Fig. 28.2). A major species difference is the presence of the major neutrophil-targeted chemokine CXCL8 in human, but not in mouse. ELR is required for function of these chemokines, which are all > 40% identical at the amino acid level (see Fig. 28.3), attract neutrophils, bind the same receptor CXCR2, and are angiogenic.⁴⁶^,⁴⁷ Of the ELR-negative CXC chemokines, only CXCL12 is angiogenic and attracts neutrophils, but by activating CXCR4.⁴⁸ The so-called IP-10 subgroup, comprised of CXCL9-11, is encoded by a distinct cluster of related genes on human chromosome 4q21. Members share the receptor CXCR3, but are angiostatic, not angiogenic.⁴⁶^,⁴⁹

The CC group is comprised of multiple subgroups. The two largest are the monocyte chemoattractant protein and macrophage inflammatory protein (MIP) clusters on human chromosome 17q12, which typically chemoattract monocytes/macrophages, plus other differential cell targets⁵⁰ (see Fig. 28.3). CC chemokines may also be subclassified by the presence or absence of additional cysteines (see Fig. 28.4). Although in separate groups as defined by cysteine motifs, CXCL16 and CX3CL1 also form a unique multimodular subgroup⁵¹^,⁵² (see Fig. 28.4). In addition to a typical chemokine domain, both these chemokines have a mucin-like stalk, a transmembrane domain, and a C-terminal cytoplasmic module, and can exist as a membrane-bound or shed form, mediating direct G protein-independent cell-cell adhesion and chemotaxis, respectively.⁵³ The discovery of CXCL17 by sequence threading techniques, not by primary sequence homology, provides proof of principle that the boundaries of chemokine sequence space could expand substantially as protein structures are determined or modeled.⁵⁴

All chemokines fold into a highly conserved and compact three-dimensional structure. Disulfide bonds link Cys-1 to Cys-3 and Cys-2 to Cys-4.⁴⁵^,⁵⁵ The core, which contains three &bgr; sheets arranged in the shape of a Greek key, is overlaid by a C-terminal &agr;-helical domain and is flanked by an N-terminal domain that lacks order (see Fig. 28.4). Forced chemokine monomers are active,⁵⁶^,⁵⁷ but dimer and tetramer structures may occur, and complex quaternary structures bound to glycosaminoglycans (GAGs) on the surface of cells may be important for function in vivo.⁴^,⁵⁸^,⁵⁹^,⁶⁰^,⁶¹^,⁶² Location of the GAG binding domains is variable and depends in part on the highly basic nature typical of most chemokines. Chemokine heterodimers have been described, both CC/CC and CC/CXC, and some may form preferentially over homodimers in a GAG-dependent manner; native heterodimers have also been identified.⁶³^,⁶⁴^,⁶⁵^,⁶⁶^,⁶⁷^,⁶⁸

Chemokine Receptor Structure and Classification

Chemokine receptors are defined as mediators that activate cellular responses upon binding chemokines. All 23 known subtypes, together with receptors for the classical lipid and peptide chemoattractant receptors, are members of the rhodopsin-like seven-transmembrane (7TM) domain superfamily of GPCRs⁸^,⁴²^,⁴³ (see Fig. 28.3; www.gpcr.org/7tm). The leukocyte chemoattractant receptors comprise the second largest subfamily of GPCRs (see Fig. 28.3). Chemokine binding, membrane anchoring, and signaling domains are formed from a single polypeptide chain ˜340 to 370 aa long. CXCR4 is the only chemokine receptor, and one of only four 7TM receptors, whose structure has been determined. When bound to small molecule and cyclic
peptide antagonists, it is a dimer, in general agreement with previous biochemical data describing ligand-independent CXCR4 dimerization⁶⁹^,⁷⁰ (Fig. 28.5). Viral GPCRs have been proposed to hijack human receptors such as CXCR4 by heterodimerization.⁷¹ Evidence for homo- and heterodimerization has been reported for other chemokine receptors, such as CCR2, CCR5, and CX3CR1, but the precise physiologic forms remain undefined.⁷²^,⁷³^,⁷⁴

FIG. 28.2. Chemokine Gene Repertoires in Mouse and Human. Data are from www.ncbi.nlm.nih.gov/genome Builds 37.2 for Homo sapiens and Mus musculus. Brackets pair regions of conserved synteny between the indicated mouse and human chromosomes. Bracket labels denote chemokine groups (see text). Gene names are from the HUGO Gene Nomenclature Committee assignments. Protein names that differ from gene names are given in parentheses. Arrows, gene location and orientation relative to centromere; Mb, megabases; plasma, chemokines found at high concentrations constitutively in plasma. Other codes are in box at lower right. The figure layout is based on a previously published design and helpful advice from Hisayuki Nomiyama of Kumamoto University.

Standard receptor names are based on the property of chemokine group restriction, as follows: receptor name = ligand group root + R (for “receptor”) + number, in order of discovery. An exception is the C chemokine receptor XCR1, where “X” distinguishes it from CR1, the previously assigned name for complement receptor 1. For consistency, the XCR1 ligands are named XCL1 and XCL2. All but six chemokines have had their receptors identified (see Table 28.3). Chemokines have unique receptor specificity profiles, and chemokine receptors have unique chemokine specificity profiles. Ligands that share a common receptor interact with the same receptor chain, in contrast to many other types of cytokines that share receptors via specialized subunits.⁷⁵ Pharmacologically, almost all chemokines are agonists, but a few are agonists at one receptor and antagonists at another (see Table 28.3).

FIG. 28.3. Chemokine and Chemokine Receptor Family Trees in Mouse and Human. Branch distances are calibrated by the bar at the bottom of each tree. The dendrograms were created in collaboration with Steven Tsang, National Institute for Allergy and Infectious Diseases.

Atypical Chemokine System Components

There are at least six categories of atypical chemokine system components. The first includes the chemokines and 7TM chemokine receptors encoded by poxviruses and herpesviruses, the probable result of “molecular piracy” of their hosts.²⁹^,⁷¹^,⁷⁶^,⁷⁷ This group includes chemokine scavengers and chemokine receptor antagonists thought to function in immune evasion; additional functions include modulation of host cell proliferation and movement. The second is a heterogeneous group of host 7TM chemokine-binding proteins that signal anomalously or not at all.⁷⁸^,⁷⁹^,⁸⁰^,⁸¹ Included in this group are Duffy antigen receptor for chemokines (DARC), CXCR7, CCBP2 (also known as D6), CCRL1, and CCRL2. CXCR7, DARC, and CCBP2 are the best studied. CXCR7 has been included in the standard nomenclature system due to evidence of &bgr;-arrestin- but not G-protein-mediated signaling.⁸² Its functions will be discussed in a later section with CXCR4, with which it shares a common ligand, CXCL12. DARC is nonsignaling and is expressed on red cells, lymphatic endothelium, and cerebellar Purkinje cells, but not

leukocytes. It is an exception to the ligand rule as it binds many, but not all, CC and CXC chemokines.⁸³ DARC mediates transcytosis of chemokines across endothelial cells⁸⁴ and modulates inflammatory responses.⁸⁵ CCBP2 is expressed on leukocytes but at low levels. More prominently expressed on placental trophoblasts⁸⁶ and lymphatic endothelium,⁸⁷ CCBP2 scavenges inflammatory CC chemokines by a process of rapid internalization and recycling to plasma membrane,⁸⁸ and affects embryo survival, inflammatory responses, immune activation, and antimicrobial resistance in infection and cancer models.⁸⁹ One theory is that nonsignaling chemokine binding proteins function as antiinflammatory chemokine buffers, and accordingly they have been referred to as chemokine scavengers, interceptors, and silent or decoy receptors. A third category includes structurally unique broad-spectrum anti-inflammatory chemokine
binding proteins encoded by microbes, including several herpesviruses and poxviruses,⁹⁰ as well as Schistosoma mansoni⁹¹ and ectoparasitic ticks.⁹²

TABLE 28.1 Chemokine Nomenclature

			Accession Number
Standard Name	Common Aliases	Other Names	Human	Mouse
CXCL1	GRO&agr;, MGSA Mouse: KC	SCYB1, NAP-3, FSP, GRO1, N51	P09341	P12850
CXCL2	Gro&bgr;; MIP-2&agr; Mouse: MIP-2	SCYB2, CINC-2a, GRO2, MGSA-&bgr;	P19875	P10889
CXCL3	Gro&ggr;, MIP-2&bgr;,	CINC-2b, GRO3, SCYB3 Mouse: Dcip1	P19876	Q6W5C0
CXCL4	Platelet factor-4	SCYB4	P02776	Q9Z126
CXCL4L1	PF4V1	CXCL4V1,SCYB4V1,	P10720
CXCL5	ENA-78 Mouse: LIX	SCYB5	P42830	P50228
CXCL6	GCP-2	SCYB6, CKA-3	P80162	NA
CXCL7	NAP-2	THBGB1; PBP => CTAP-III => &bgr;-TG => NAP-2^a SCYB7, &bgr;-TG1, Beta-TG, CTAP3, LA-PF4, LDGF, MDGF	P02775	Q9EQI5
CXCL8	IL-8	3-10C, AMCF-I, &bgr;-ENAP, GCP-1, K60, LECT, LUCT, MDNCF, MONAP, NAF, NAP-1, SCYB8, TSG-1	P10145	NA
CXCL9	Mig	SCYB9, crg-10, Humig Mouse: CRG-1	Q07325	P18340
CXCL10	&ggr;IP-10	SCYB10, C7, crg-2, IFI10, mob-1 Mouse: CRG-2	P02778	P17515
CXCL11	I-TAC	IP9, H174, SCYB11	O14625	Q8R392
CXCL12	SDF-1&agr;	SDF-1&bgr;^b, PBSF, SCYB12, TPAR1, TLSF	P48061	P40224
CXCL13	BLC	BCA-1, SCYB13, ANGIE, ANGIE2, BLR1L	O43927	O55038
CXCL14	BRAK	bolekine, SCYB14, BMAC, Kec, KS1, MIP-2&ggr;, NJAC	O95715	Q6AXC2
Cxcl15	Lungkine		NA	Q9WVL7
CXCL16	SR-PSOX		Q9H2A7	Q8BSU2
CXCL17		Dcip1, DMC, UNQ473, VCC1	Q6UXB2	Q8R3U6
CCL1	I-309	P500, SIS&egr; Mouse: TCA-3	P22362 P10146
CCL2	MCP-1 Mouse: JE	MCAF, HC11, SCYA2, SMC-CF, GDCF-2	P13500 P10148
CCL3	MIP-1&agr;	MIP-1&agr;S, LD78&agr;, GOS19-1; PAT 464.1; TY-5; SIS&agr;	P10147	P10855
CCL3L1		MIP-1&agr;P, LD78&bgr; SCYA3L, SCYA3L1, G0S19-2; PAT 464.2	P16619	P10855
CCL3L3		MGC12815	P16619
CCL4	MIP-1&bgr;	ACT-2, PAT 744, H400; SIS-&ggr;, LAG-1, HC21, G-26, MAD-5, AT744.1	P13236	P14097
CCL4L1		AT744.2, LAG-1	Q8NHW4	NA
CCL4L2			Q8NHW4	NA
CCL5	RANTES	SIS-&dgr;, MGC17164, p288, TCP228	P13501	P30882
Ccl6	C10, MRP-1		NA	P27784
CCL7	MCP-3	NC28, FIC Mouse: MARC	P80098	Q03366
CCL8	MCP-2	HC14	P80075	Q9Z121
Ccl9	MRP-2, MIP-1&ggr;	CCF18	NA	P51670
CCL10 (reserved)			NA	NA
CCL11	Eotaxin	SCYA11, eotaxin-1, MGC22554	P51671	P48298
Ccl12	Mcp-5		NA	Q62401
CCL13	MCP-4	Ck&bgr;10; NCC-1, MGC17134, SCYL1, SCYA13	Q99616	NA
CCL14	HCC-1	CC-1, NCC-2, CCCK-1/CCCK-3, Ck&bgr;1, MCIF, HCC-3, SCYL2, SCYA14	Q16627	NA
CCL15	HCC-2	Leukotactin-1 (Lkn-1), MIP-5, CC-2, NCC-3, MIP-1&dgr;, HMRP-2B, SCYL3, SCYA15	Q16663	NA
CCL16	HCC-4	LEC, NCC-4, LMC, monotactin-1 (Mtn-1), LCC-1, ILINCK, CK&bgr;12, SCYL4, SCYA16	O15467	NA
CCL17	TARC	STCP-1, SCYA17	Q92583	Q9WUZ6
CCL18	PARC	DC-CK-1, MIP-4, AMAC-1, ck&bgr;7, SCYA18	P55774	NA
CCL19	ELC	MIP-3&bgr;, Exodus-3, ck&bgr;11, SCYA19	Q99731	O70460
CCL20	MIP-3&agr;, LARC	Exodus-1, CK&bgr;4, SCYA20 Mouse: ST38	P78556	O89093
CCL21	SLC	6Ckine, exodus-2, TCA4, ck&bgr;9, ECL, SCYA21	O00585	P84444
CCL22	MDC	STCP-1, SCYA22 Mouse: abcd-1, dc/b-ck	O00626	O88430
CCL23	MPIF-1	MIP-3, ck&bgr;8-1, SCYA23	P55773	NA
CCL24	Eotaxin-2	MPIF-2, ck&bgr;6, SCYA24	O00175	Q9JKC0
CCL25	TECK	ck&bgr;15, SCYA25	O15444	O35903
CCL26	Eotaxin-3	MIP-4&agr;, IMAC, N1, TSC-1, SCYA26	Q9Y258	Q5C9Q0
CCL27	CTACK	ILC, SCYA27, ESkine, PESKY, skinkine mouse: ALP	Q9Y4X3	Q9Z1X0
CCL28	MEC	SCYA28, CCK1	Q9NRJ3	Q9JIL2
XCL1	Lymphotactin &agr;	SCM-1&agr;, ATAC^a, SCYC1, LPTN	P47992	P47993
XCL2	Lymphotactin &bgr;	SCM-1&bgr;, ATAC, SCYC2	Q9UBD3	NA
CX₃CL1	Fractalkine	SCYD1, ABCD-3, C3Xkine, CXC3, CXC3C, Mouse: neurotactin	P78423	O35188
6Ckine, chemokine with 6 cysteines; AMAC, alternative macrophage activation-associated CC-chemokine; ATAC, activation-induced, chemokine-related molecule exclusively expressed in CD8+ T lymphocytes; BCA-1, B cell activating chemokine-1; BLC, B lymphocyte chemoattractant; BRAK, Breast and kidney chemokine; CC-#, CC chemokine-#; ckb#, CC chemokine #; CD, cluster of differentiation; CTACK, cutaneous T cell attracting chemokine; CTAP, connective tissue activating peptide; DC, dendritic cell; dc/b-ck, dendritic cell beta chemokine-1; ELC, Epstein-Barr virus-induced receptor ligand chemokine; ENA-78, epithelial cell-derived neutrophil-activating factor, 78 amino acids; FIC, fibroblast inducible cytokine; GCP-#, granulocyte chemoattractant protein-#; Gro, growth-related oncogene; HCC-#, hemofiltrate CC chemokine-#; IL-8, interleukin-8; IP-10, interferon-inducible protein-10; I-TAC, IFN-inducible T cell alpha chemoattractant; LARC, liver and activation-related chemokine; LCC, liver CC chemokine; LEC, liver expressed chemokine; LMC, lymphocyte and monocyte chemoattractant CC chemokine; LYNAP, lymphocyte-derived neutrophil-activating peptide; MARC, mast cell activation related chemokine; MCAF, monocyte chemoattractant and activating factor; MCIF, macrophage colony inhibitory factor; MCP, monocyte chemoattractant protein; MDC, macrophagederived chemokine; MDNCF, monocyte-derived neutrophil chemotactic factor; MEC, mucosa-associated epithelial cell chemokine; MGSA, melanoma growth-stimulatory activity; Mig, monokine induced by gamma interferon; MIP, macrophage inflammatory protein; MPIF-#, myeloid progenitor inhibitory factor-#; MRP-#, MIP-related protein-#; Mtn-1, monotactin-1; NAF, neutrophil-activating factor; NAP-#, neutrophil-activating protein-#; NCC-#, novel CC chemokine-#; PARC, pulmonary and activation-regulated chemokine; PBP, platelet basic protein; PBSF, pre B cell stimulatory factor; PF4, platelet factor 4; RANTES, regulated upon activation normal T cell-expressed and secreted; SCM, single cysteine motif-1; SCY#, small cytokine #; SDF-1, stromal cell-derived factor-1; SIS-#, small inducible secreted protein-#; SLC, secondary lymphoid tissue chemokine; SR-PSOX, scavenger receptor for phosphatidylserine and oxidized lipoprotein; TARC, thymus and activation-related chemokine; TCA-#, T-cell activation protein-#; TECK, thymus expressed chemokine; TG, thromboglobulin; TLSF, thymic lymphoma cell stimulating factor; TPAR, TPA repressed protein.
^aSequential N-terminal truncation of PBP produces the chemokines shown. Only NAP-2 has leukocyte chemoattractant activity, specifically for neutrophils.
^bSDF-1&agr; and &bgr; are splice variants of the same human gene.

TABLE 28.2 Chemokine Receptor Nomenclature

			Accession Number
Name	CD#	Previous Names	Human	Mouse
CXCR1	CD181	IL8R_A, IL-8R-I, CDw128a, CKR-1, IL-8R&agr;	P25024	Q810W6
CXCR2	CD182	IL8R_B, IL-8R-II, CMKAR2, IL-8R&bgr;	P25025	P35343
CXCR3	CD183	IP10/Mig R, GPR9, CKR-L2, CMKAR3, IP10-R, MigR	P49682	O88410
CXCR4	CD184	HUMSTSR, LESTR, fusin, HM89, LCR1, NPYR, D2S201E, D2S201E, fusin, HSY3RR, NPY3R,	P61073	P70658
CXCR5	CD185	BLR-1, MDR15	P32302	Q04683
CXCR6	CD186	BONZO, STRL33, TYMSTR	O00574	Q9EQ16
CXCR7		RDC1,GPR159, CMKOR1	P25106	P56485
CCR1	CD191	CKR1, CC CKR1, MIP-1&agr;/RANTES, CMKBR1	P32246	P51675
CCR2	CD192	CKR2, CC CK2, CC CKR2, MCP-1, CMKBR2 CC-CKR-2, FLJ78302, MCP-1-R	P41597	P51683
CCR3	CD193	CKR3, CC CKR3, Eotaxin receptor, CMKBR3	P51677	P51678
CCR4	CD194	CKR4, CC CKR4, K5-5, CMKBR4, CHEMR1	P51679	P51680
CCR5	CD195	CKR5, CC CKR5, ChemR13, CMKBR5, IDDM22	P51681	P51682
CCR6	CD196	GPR-CY4, CKR-L3, STRL-22, DRY-6, DCR2, BN-1, GPR29, CMKBR6	P51684	O54689
CCR7	CD197	EBI-1, BLR-2, CMKBR7	P32248	P47774
CCR8	CDw198	TER1, CKR-L1, GPR-CY6, CMKBR8, CKR-L1	P51685	P56484
CCR9	CDw199	GPR28, GPR 9-6	P51686	Q9WUT7
CCR10		GPR2	P46092	Q9JL21
XCR1		GPR5	P46094	Q9R0M1
CX₃CR1		Fractalkine receptor, GPR13, V28, CMKBRL1, CCRL1, CMKDR1	P49238	Q9Z0D9
DARC	CD234	Duffy; glycoprotein D, CCBP1, Dfy, GPD, FY	Q16570	Q9QUI6
CCBP2		D6, CCR9 (unofficial), CCR10 (unofficial)	O00590	Y12879
CCRL1		CCX-CKR, CCBP2, CCR11, PPR1, VSHK1	Q9NPB9	Q924I3
CCRL2		CKRX, CRAM-A, L-CCR, CRAM-B, HCR, CCR11 (unofficial)	O00421	O35457

FIG. 28.4. Chemokine Classification, Nomenclature, and Structure. A: Chemokine groups are defined by the number and arrangement of conserved cysteines, as shown. Brackets link cysteines that form disulfide bonds. ELR, glu-leu-arg; X, an amino acid other than cysteine. The underscore is a spacer used to optimize the alignment. The N- and C– termini can vary considerably in length (not illustrated). For the molecules with four cysteines, there are approximately 24 amino acids between Cys-2 and Cys-3 and 15 amino acids between Cys-3 and Cys-4. At right is listed the nomenclature system. B: The chemokine fold illustrated by monomeric CXCL8. C: Model of transmembrane chemokine CX3CL1. See text for details.

TABLE 28.3 Chemokine Specificities for Human Seven-Transmembrane Chemokine Receptors

CXC

CX3C

Atypical

CXCR1

CXCR2

CXCR3

CXCR4

CXCR5

CXCR6

CCR1

CCR2

CCR3

CCR4

CCR5

CCR6

CCR7

CCR8

CCR9

CCR10

XCR1

CX3CR1

DARC

CCBP2

CCRL1

CCRL2

CXCR7

CXC

CXCL1/Gro&agr;

CXCL2/Gro&bgr;

CXCL3/Gro&ggr;

CXCL4/PF-4

CXCL4L1

CXCL5/ENA-78

CXCL6/GCP-2

CXCL7/NAP-2

CXCL8/IL-8

CXCL9/Mig

CXCL10/&ggr;IP-10

CXCL11/I-TAC

CXCL12/SDF-1

CXCL13/BCA1

CXCL14/BRAK

*Cxcl15/lungkine

CXCL16

CXCL17

CCL1/I-309

CCL2/MCP-1

CCL3/MIP-1&agr;

CCL4/MIP-1&bgr;

CCL5/RANTES

*Ccl6/MRP-1

CCL7/MCP-3

CCL8/MCP-2

*Ccl9/MRP-2

CCL11/eotaxin

*Ccl12/MCP-5

CCL13/MCP-4

CCL14a/HCC-1

CCL14b/HCC-3

CCL15/HCC-2

CCL16/HCC-4

CCL17/TARC

CCL18/PARC

CCL19/ELC

CCL20/LARC

CCL21/SLC

CCL22/MDC

CCL23/MPIF-1

CCL24/eotaxin-2

CCL25/TECK

CCL26/eotaxin-3

CCL27/CTACK

CCL28/MEC

XCL1/lymphotactin &agr;

XCL2/lymphotactin &bgr;

CX3C

CX3CL1/fractalkine

MCP, monocyte chemoattractant protein; NAP, neutrophil-activating protein.

TABLE 28.4 Chemokine Receptor Distribution on Some Hematopoietic Cells

CXC

CX3C

Atypical

CXCR1

CXCR2

CXCR3

CXCR4

CXCR5

CXCR6

CCR1

CCR2

CCR3

CCR4

CCR5

CCR6

CCR7

CCR8

CCR9

CCR10

XCR1

CX3CR1

DARC

CCBP2

CCRL1

CCRL2

CXCR7

CD34+ HSC

Erythrocytes

Megakaryocytes

Platelets

Granulocytes

Neutrophils

Eosinophils

Basophils

Mononuclear Phagocytes

CD14+ Monocytes

CD16+ Monocytes

Immature DC

Mature DC

NK Cells

CD56^dim CD16+ NK

CD56^bright CD16- NK

NK-T cells

CD4 NK-T

CD8 NK-T

CD4-CD8- NK-T

B cells

Marginal Zone B Cells

Plasma cells

IgA Ab-secreting cells

T cells

Naïve T cells

Follicular help T cells

Central memory T cells

Effector memory T cells

Th1 Effector T cells

Th2 Effector T cells

&agr;4&bgr;7+ Gut-homing memory T cells

CLA+ Skin-homing memory T cells

CD4+ CD25+Foxp3 + Regulatory T cells

CD, cluster of differentiation; DC, dendritic cell; Ig, immunoglobulin; HSC, hematopoietic stem cell.

FIG. 28.5. Chemokine Receptor Structure. A: Structure of CXCR4 bound to ligand IT1t, at left⁷⁰; CXCR4 dimer, at right, illustrating the dimer contact surface at the bottom right.⁷⁰ B: CCL2 monomer docking to model of CCR2.¹³⁵

The fourth group of atypical components includes endogenous nonchemokine ligands that bind chemokine receptors. Examples include aminoacyl transfer ribonucleic acid synthetases, some of which function as autoantigens in autoimmune disorders⁹³^,⁹⁴; the antimicrobial peptide &bgr; defensin 2, which activates CCR6 on immature dendritic cells (DCs), possibly linking innate to adaptive immunity⁹⁵; and cluster of differentiation (CD)82 (KAI1), a tetraspanin expressed on leukocytes and cancer cells that binds to endothelial cell-expressed DARC, triggering senescence in tumor cells and suppression of metastasis.⁹⁶ The fifth group includes chemokines that bind scavenger receptor ligands, such as oxidized LDL, through their receptor binding domains.⁹⁷ The prototype is CXCL16, which was originally called scavenger receptor for phosphatidyl serine and oxidized LDL, or SR-PSOX. CXCL16 has also been shown to function as a transporter for CpG dinucleotides across the plasma membrane for action at toll-like receptor 9, as well as to mediate bacterial phagocytosis by antigen-presenting cells.⁹⁸

In contrast, a truncated form of CCL23, unlike full-length CCL23, has not been shown to activate any chemokine receptors, yet is an agonist at FPR2, a member of the classical chemoattractant fMet-Leu-Phe receptor subfamily.⁹⁹ Perhaps the most surprising example of an alternative chemokine receptor agonist is the tripeptide Pro-Gly-Pro, a breakdown product of collagen from extracellular matrix that is found in bronchoalveolar lavage samples from patients with chronic obstructive pulmonary disease.¹⁰⁰ This peptide shares structural homology with a domain in CXC chemokines and has been shown to recruit neutrophils via CXCR2 activation in a mouse model of lung inflammation. It has been reported to be a chemotactic agonist at CXCR2, but this has not been confirmed.¹⁰¹

A final category of atypical components includes nonchemokine ligands for chemokine receptors (agonists or antagonists) produced by pathogens. For example, HIV Tat is an antagonist at CXCR4,¹⁰² and CCR5 can be activated by viral (HIV gp120), bacterial (Mycobacterium tuberculosis Hsp70), and protozoan (Toxoplasma gondii immunophilin) factors.¹⁰³^,¹⁰⁴^,¹⁰⁵^,¹⁰⁶ M. tuberculosis Hsp70 activation of CCR5 induces DC aggregation, T-DC immune synapse formation, and effector immune responses.

Chemokine System Genes and Evolution

The chemokine system appears to have originated in teleost fish (for updates and phylogenies, see the Cytokine Family cDNA database at http://cytokine.medic.kumamoto-u.ac.jp). Comparative whole genome analysis has now shown that the chemokine repertoire differs dramatically in size throughout phylogeny with zebrafish having 63 chemokine genes compared to 48 in human.⁴⁰^,⁴¹^,¹⁰⁷^,¹⁰⁸ Only human CXCL12 and CXCL14 have unambiguous orthologues in fish, and none of the other mammalian CXC chemokines has clear orthologues in any other vertebrate class, including birds. In addition, gene copy number may differ among closely related species (see Fig. 28.2 for human and mouse) and among individuals of the same species (eg, the triplicated MIP cluster in humans). A model of chemokine system evolution has been proposed involving lineage-specific en bloc and tandem duplications with expansion and functional specialization accompanying the origin of adaptive immunity.¹⁰⁷

Chemokine genes typically are ˜4 kb long and usually have four exons in the case of CXC chemokines and three exons in the case of CC chemokines. The promoter is immediately upstream of exon 1, which encodes a leader sequence and a few amino acids of the mature peptide. Alternative
splicing has been reported for several chemokines, but the significance is not well defined. CCL14 and CCL15 are an exceptional example of neighboring genes that give rise to a family of mono- and bicistronic transcripts.¹⁰⁹

The 23 chemokine receptor genes can be divided by chromosomal location into three groups: a large cluster on human chromosome 3p21-23 including multiple CCRs, CX3CR1, XCR1, and CXCR6, plus CCBP2 and CCRL1; CXCR1, CXCR2, and one receptor pseudogene clustered on 2q34-q35; and the rest unclustered. With the exception of CCR9, genes in the two clustered groups lack introns in the open reading frame (ORF) but have at least one and as many as 10 introns separating the promoter from the ORF. In contrast, an intron divides the N-terminus of the majority of unclustered receptor genes (CXCR3, 4, and 5; CCR6 and 10). Several of these undergo alternative splicing but the products appear to function similarly. CCR2 undergoes alternative splicing of a virtual intron in the C-terminal region of the ORF, but the two products have similar function and CCR2B appears to be the major expressed form.

With regard to horizontal evolution, variation in gene copy number among individuals has been observed for only a few chemokines. CCL3 is an example of this in human,¹¹⁰ and the plt locus (paucity of lymph node T cells), which will be discussed in a later section, is an immunologically important example in mouse¹¹¹ (see Fig. 28.2). In contrast, variation in gene sequence is common among individuals for most chemokine and chemokine receptors. However, the degree of polymorphism varies greatly among different genes. The most extreme and important example is the CCR2-CCR5 locus in human, in which combinations of common dimorphic single nucleotide polymorphisms in the CCR5 promoter with a single nucleotide polymorphism in the ORF of CCR2 named CCR2 V64I and a 32 base pair deletion in the ORF of CCR5 named CCR5&Dgr;32 together form at least eight distinct haplotypes and in some cases affect HIV disease susceptibility.¹¹²^,¹¹³^,¹¹⁴

TABLE 28.5 Sources and Main Immunologic Functions of Human CXC, CX3C, and C Chemokines

Chemokine	Main Source	Main Immunologic Roles
CXCL1	Inducible in most hematopoietic and tissue cells	Neutrophil trafficking
CXCL2	Many tumors Neutrophil	trafficking
CXCL3		Neutrophil trafficking
CXCL4	Preformed in platelets	Procoagulant
CXCL4L1	Preformed in platelets	ND
CXCL5	Induced in epithelial cells of gut and lung; N, Mo, Plts, EC	Neutrophil trafficking
CXCL6	Induced in lung microvascular EC; Mo; alveolar epithelial cells, mesothelial cells, EC and M&PHgr;	Neutrophil trafficking
CXCL7	Preformed in platelets	Neutrophil trafficking
CXCL8	Induced in most cell types	Neutrophil trafficking
CXCL9	Induced in PMN, M&PHgr;, T cells, astrocytes, microglial cells, hepatocytes, EC, fibroblasts, keratinocytes, thymic stromal cells	Th1 response
CXCL10	Induced in ECs, Mo, keratinocytes, respiratory & intestinal epithelial cells, astrocytes, microglia, mesangial cells, smooth muscle cells	Th1 response
CXCL11	ECs, Mo	Th1 response
CXCL12	Constitutive in bone marrow stromal cells; most tissues	Myelopoiesis HPC, neutrophil homing to BM B lymphopoiesis
CXCL13	Constitutive in follicular HEV of secondary lymphoid tissue	Naïve B- and T-cell homing to follicles B1 cell homing to peritoneum Natural Ab production
CXCL14	Constitutive in most tissues, breast and kidney tumors	Macrophage migration
(CXCL15)	Constitutive in lung epithelial cells	Neutrophil trafficking
CXCL16	Constitutive in spleen; DCs of the T zone	T-cell, NK and DC trafficking
CX3CL1	EC, neurons, Mo, DC	NK, Monocyte, M&PHgr;, and Th1 cell migration; neuroprotection
XCL1	&ggr;&dgr; epidermal T cells, NK, NK-T, activated CD8+ and Th1 CD4+ T cells	Cross presentation of antigen by XCR1+ CD8+ DC to CD8+ T cells
XCL2

Immunologic Classification of the Chemokine System

Chemokines and chemokine receptors have differential leukocyte specificity but together regulate all leukocyte subsets (Tables 28.5 and 28.6). As such, they can be loosely divided into two main functional systems, homeostatic and

inflammatory. Homeostatic chemokines are differentially and constitutively expressed primarily in specific microenvironments of primary and secondary immune organs, and coordinate migration of hematopoietic precursor cells, mature DCs, and naïve and central memory lymphocyte subsets via constitutively expressed receptors.¹¹⁵^,¹¹⁶^,¹¹⁷^,¹¹⁸^,¹¹⁹ Inflammatory chemokines are induced by noxious stimuli in tissue cells and leukocytes. Inflammatory chemokine receptors tend to be constitutively expressed on myeloid cells, natural killer (NK) cells, and effector but not naïve lymphocytes.¹²⁰^,¹²¹ Dynamic shifts in receptor expression occur during leukocyte differentiation, maturation, and activation.¹¹⁵^,¹²²^,¹²³^,¹²⁴^,¹²⁵ This is not an absolute classification, because constitutive chemokines may be further induced, and chemokines that are highly inducible in some cell types may be constitutively expressed in others. In addition, there may be differences between mouse and human leukocyte subsets. Table 28.4 condenses a small portion of this vast subject available from the literature. Additional information can be obtained from the Immunologic Genome Project (www.immgen.org

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