First printed in R&D Systems' 1998 Catalog.
Chemokines constitute a superfamily of small, inducible, secreted, proinflammatory cytokines involved in a variety of immune responses, acting primarily as chemoattractants and activators of specific types of leukocytes.1-3 Four classes of chemokines have been defined by the arrangement of the conserved cysteine (C) residues of the mature proteins: the CXC chemokines that have one amino acid (aa) residue separating the first two conserved cysteine residues; the CC chemokines in which the first two conserved cysteines residues are adjacent; the C chemokines that lack two (the first and third) of the four conserved cysteine residues; and the CX3C chemokines which have three intervening aa residues between the first two conserved cysteine residues. Within the CXC subfamily, the chemokines can be further divided into two groups: 1) having the characteristic three aa sequence ELR (glutamic acid-leucine-arginine) motif immediately preceding the first conserved cysteine residue near the amino terminus; and 2) lacking such an ELR domain. During the past two years, many new chemokines, especially CC chemokines, have been discovered from EST databases as a result of bioinformatics.
Many of the ELR containing CXC chemokines have been shown to be chemotactic for neutrophils while non-ELR CXC chemokines are chemotactic for lymphocytes. By contrast, the CC chemokines were found to be chemotactic for monocytes, T lymphocytes, B lymphocytes, dendritic cells, natural killer cells, eosinophils and basophils, but not neutrophils. The unique C chemokine lymphotactin was reported to be chemotactic for lymphocytes. The sole CX3C chemokine (fractalkine), a type 1 membrane protein containing a chemokine domain tethered on a long mucin-like stalk, has been found to trigger the adhesion of T cells and monocytes.4, 5 The activities of many of the chemokines recently discovered are just being elucidated.
|Figure 1: Chemokine Recruitment Cascade.|
Genes for many CXC and CC chemokines have been found to be clustered. Most of the CXC chemokines genes have been mapped to human chromosome 4q, and those for many CC chemokines on human chromosome 17q (mouse chromosome 11). Exceptions to these clusters include SDF-1 (a CXC chemokine) on human chromosome 10; MIP-3 alpha, MIP-3 beta, 6Ckine, and TARC (CC chemokines) on human chromosome 2, 9, 9, and 16, respectively. The clustering of chemokine genes suggests that many chemokine family members arose through gene duplication and subsequent divergence. The gene for lymphotactin (C chemokine) has been localized to chromosome 1 in both human and mouse while that for fractalkine (CX3C chemokine) has been mapped to human chromosome 16q and mouse chromosome 8.
Chemokines mediate their activities by binding to target cell surface chemokine receptors that belong to the large family of G protein-coupled, seven transmembrane domain receptors.1, 2, 6 To date, four CXC chemokine receptors (CXCR-1 through 4), at least eight CC chemokine receptors (CCR-1 through 8) and one CX3C chemokine receptor (CX3CR) have been cloned and characterized. The various chemokine receptors are known to exhibit overlapping ligand specificities. In addition to chemokine receptors with known ligand(s), numerous orphan chemokine receptors for as yet unidentified ligands have been reported. In addition, the Duffy blood group antigen (DARC) has been shown to be a chemokine receptor that can bind selected CXC and CC chemokines. Great interest has been generated by the finding that HIV viruses use some chemokine receptors as co-receptors for entry into cells.7-9
A number of virally encoded chemokines (e.g., three HHV-8 encoded chemokines, vMIP-I, vMIP-II, vMIP-III) and chemokine receptors (e.g., HHV-8 ORF74 encoded chemokine receptor GPCR) have been identified.10-12 Viral chemokines and chemokine receptors apparently are pirated from cellular genes by the viruses. vMIP-II has been shown to act as a potent and efficient antagonist of chemotaxis by chemokines and binds with high affinity to a number of both CC and CXC chemokine receptors. The viral chemokine receptor GPCR, similar to DARC, has been shown to bind both CXC and CC chemokines.
Fractalkine, also named neurotactin, is a chemokine recently identified through bioinformatics.13, 14 Fractalkine has a unique CX3C cysteine motif near the amino-terminus and is the first member of a fourth branch of the chemokine superfamily. Unlike other known chemokines, fractalkine is a type 1 membrane protein containing a chemokine domain at the amino terminus tethered on a long mucin-like stalk. Human fractalkine cDNA encodes a 397 aa residue membrane protein with a 24 aa residue predicted signal peptide, a 76 aa residue chemokine domain, a 241 aa residue stalk region containing 17 degenerate mucin-like repeats, a 19 aa residue transmembrane segment and a 37 aa residue cytoplasmic domain. Mouse fractalkine cDNA also encodes a 395 aa residue membrane protein (signal peptide, residues 1-21; chemokine domain, residues 22-105, transmembrane domain, residues 339-358). The extracellular domain of fractalkine has been shown to be released into the supernatants of transfected cells as a 95 kDa glycoprotein, possibly by proteolysis at the dibasic cleavage site proximal to the membrane, to generate soluble fractalkine.
Human fractalkine mRNA expression is most abundant in the brain and heart, but is also present at lower levels in other tissues tested except peripheral blood where most chemokines are highly expressed. Mouse fractalkine mRNA is also principally detected in brain and to a lesser extent in other tissues. The expression of fractalkine in microglia, endothelial and fibroblastic cells have been reported to be upregulated by inflammatory signals. The gene for human fractalkine has been mapped to chromosome 16q.
Cell-surface fractalkine has been shown to promote adhesion of monocytes and T cells to cells expressing the membrane protein. Adhesion was found to be a function of the chemokine domain since the recombinant soluble chemokine domain can inhibit this effect. Both recombinant human soluble fractalkine containing the entire extracellular domain, or only the chemokine domain, have been reported to chemoattract monocytes and lymphocytes but not neutrophils in vitro. In contrast, the mouse fractalkine chemokine domain was reported to be chemotactic for neutrophils both in vitro and in vivo. Mouse recombinant soluble fractalkine containing the entire extracellular domain was also shown to chemoattract neutrophils in vivo but not in vitro. The basis for these differences is not yet understood. A receptor for fractalkine, CX3CR has recently been identified and cloned.15
MIP-1 gamma also named CCF18, is a mouse CC chemokine independently cloned by two different groups from a macrophage cDNA library or from a cDNA library of an IL-3-dependent pro-B cell line, Ba/F3.16-18 Mouse MIP-1 gamma cDNA encodes a 122 aa residue precursor protein with a predicted 21 (22) aa residue hydrophobic signal peptide that is cleaved to generate the 101 (100) aa residue mature secreted protein. Among CC chemokines family members, mouse MIP-1 gamma shares approximately 45%, 24% and 20% aa sequence identity with mouse C10, mouse MIP-1 alpha and mouse MIP-1 beta, respectively. The gene for MIP-1 gamma has been mapped to mouse chromosome 11, where the genes for many mouse CC chemokines are clustered. Unlike chemokines such as MIP -1 alpha and MIP-1 beta, whose expression is induced by inflammatory stimuli, MIP-1 gamma is constitutively expressed in macrophage, dendritic cells and myeloid cell lines. Relatively high concentrations (1 Âµg/mL) of MIP-1 gamma has been detected in the serum. Recombinant MIP-1 gamma has also consistently been shown to induce chemotaxis and Ca2+ flux in CD4+ or CD8+ T cells and in both activated and unactivated T cells.
MIP-3 alpha, also known as LARC (Liver and Activation-regulated Chemokine) and Exodus-1, is one of many CC chemokines recently identified through bioinformatics.19-21 MIP-3 alpha cDNA encodes a 96 aa residue precursor protein with a 26 aa residue signal peptide that is predicted to be cleaved to form the 70 aa residue mature secreted protein. MIP-3 alpha is distantly related to other CC chemokines (20 - 28% aa sequence identity) and the gene for MIP-3 alpha has been mapped to chromosome 2q33 - q37 rather than chromosome 17 where many of the CC chemokines are clustered. MIP-3 alpha mRNA is predominantly expressed in liver, fetal lung, lymph nodes, appendix, peripheral blood lymphocytes and several cell lines. The expression of MIP-3 alpha is strongly up-regulated by inflammatory signals and down-regulated by the anti- inflammatory cytokine IL-10.
Recombinant MIP-3 alpha has been shown to be chemotactic for lymphocytes but not for monocytes. Synthetic Exodus-1, which differs from recombinant MIP-3 alpha by having three additional aa residues at the amino terminus, was also reported to inhibit proliferation of myeloid progenitors in colony formation assays. MIP-3 alpha has now been shown to be a unique functional ligand for CCR-6 (previously referred to as the GPR-CY4, CKR-L3, or StrL22 orphan receptor), a CC chemokine receptor that is highly expressed in human dendritic cells derived from CD34+ cord blood precursors.22-24 In addition to dendritic cells, CCR-6 mRNA has also been detected in lymphocytes (CD4 and CD8 T cells and B cells), but not in natural killer cells, monocytes and granulocytes.
6Ckine is a CC chemokine recently discovered independently from the EST database by three groups. 6Ckine, also named SLC (secondary lymphoid-tissue chemokine) and Exodus-2, shows 21 - 33% identity to other CC chemokines.25-27 6Ckine contains the four conserved cysteines characteristic of beta chemokines plus two additional cysteines in its unusually long carboxyl-terminal domain. Human 6Ckine cDNA encodes a 134 aa residue, highly basic, precursor protein with a 23 aa residue signal peptide that is cleaved to form the predicted 111 aa residues mature protein. Mouse 6Ckine cDNA encodes a 133 aa residue protein with 23 residue signal peptide that is cleaved to generate the 110 residue mature protein. Human and mouse 6Ckine is highly conserved, exhibiting 86% aa sequence identity. 6Ckine is constitutively expressed at high levels in lymphoid tissues such as lymph nodes, spleen, and appendix. In mouse, high levels of 6Ckine mRNA are also detected in the lung. The gene for human 6Ckine has been localized at human chromosome 9p13 rather than chromosome 17, where the genes of many human CC chemokines are clustered. The 6Ckine gene location is within a region of about 100 kb as the gene for MIP-3 beta/ELC, another recently identified CC chemokine.
Unlike most CC chemokines, 6Ckine is not chemotactic for monocytes. Recombinant mouse 6Ckine is chemotactic for thymocytes and activated T cells in vitro. Recombinant human 6Ckine has been shown to be chemotactic for some human T cell lines, resting PBL and cultured T cells expanded with PHA and IL-2. 6Ckine has also been reported to inhibit hemopoietic progenitor colony formation in a dose-dependent manner. 6Ckine acts via a class of as yet unidentified CC receptor on both T cells and B cells that are not shared by any other CC chemokines tested so far.
MIP-3 beta, also known as ELC (EBI-1 Ligand Chemokine), is another CC chemokine identified via bioinformatics.28, 29 MIP-3 beta cDNA encodes a 98 aa residue highly basic precursor protein with a predicted 21 aa residue signal peptide that is cleaved to form the 77 aa residue mature secreted protein. MIP-3 beta is distantly related to other CC chemokines (29 - 30% aa sequence identity) and the gene for MIP-3 beta has been mapped to chromosome 9p13 rather than chromosome 17, within a 100 kb of the gene for 6Ckine. MIP-3 beta is constitutively expressed at high levels in thymus, lymph nodes, appendix and tonsil. In addition MIP-3 beta mRNA is also expressed by activated monocytes but not by dendritic cells or PBMNC. The expression of MIP-3 beta is down-regulated by the anti-inflammatory cytokine, IL-10.
Recombinant MIP-3 beta has been shown in vitro to be chemotactic for cultured human lymphocytes expanded with or without IL-2. Similar to 6Ckine, MIP-3 beta is not chemotactic for monocytes. MIP-3 beta has been shown to be a unique functional ligand for CCR-7 [previously referred to as the Epstein-Barr virus-induced gene 1 (EBI-1) orphan chemokine receptor], a CC chemokine receptor that is expressed in various lymphoid tissues and activated B and T cells. EBI-1 is strongly up-regulated in B cells infected with Epstein-Barr virus and T cells infected with herpesvirus 6 or 7.
GCP-2 (granulocyte chemotactic protein-2) is a CXC chemokine initially isolated as a neutrophil chemoattractant from the MG-63 osteosarcoma cell line.30 Among, human CXC chemokines, GCP-2 is most closely related to ENA-78 (78% aa sequence identity in the mature peptide region and 86% identity in the signal sequence). The structure and sequence of the genes for human GCP-2 and ENA-78 also exhibit close similarity suggesting the two genes may have originated from a recent gene duplication. liX (LPS-induced CXC chemokine) was initially cloned as a gene induced by LPS in fibroblasts.31 A mouse protein with a sequence corresponding to the predicted liX protein sequence was recently purified and designated mouse GCP-2 based on its aa sequence similarity (60% sequence identity) to human GCP-2. However, mouse GCP-2/liX is also 54% identical with human ENA-78 at the aa sequence level.
Human GCP-2 cDNA encodes a propeptide of 114 aa residues with a predicted 37 aa residue signal peptide and a 77 aa residue mature protein.32 Compared to the 75 aa residue natural protein isolated from MG-63 conditioned media, the predicted mature human GCP-2 contains two additional aa residues at the carboxyl terminus. Human GCP-2 is a primary response gene whose induction by cytokines is attenuated by dexamethasone.
Mouse GCP-2/liX cDNA encodes a 132 aa residue propeptide with a predicted 40 aa residue signal peptide and 92 aa residue mature protein.31, 33 Natural mouse GCP-2/liX purified from fibroblasts and epithelial cells contains multiple amino-terminally processed forms of the protein, all of which include the ELR motif. The shorter forms of the natural protein were reported to be more active than the longer forms. Mouse GCP-2/liX is induced by LPS in fibroblasts, but not in macrophages, and is down-regulated by dexamethasone.
Human GCP-2 and mouse GCP-2/liX have been shown to chemoattract and activate neutrophils, but not eosinophils and monocytes. It is likely that GPC-2 activities are mediated via the human or mouse CXCR-2.
TARC (thymus and activation-regulated chemokine ) is a CC chemokine recently identified using a signal sequence trap cloning method.34-36 TARC cDNA encodes a highly basic 94 aa residue precursor protein with a 23 aa residue signal peptide that is cleaved to generate the 71 aa residue mature secreted protein. Among CC chemokine family members, TARC has approximately 24-29% aa sequence identity with RANTES, MIP-1 alpha, MIP-1 beta, MCP-1, MCP-3, and I-309. The gene for human TARC has been mapped to chromosome 16q13 rather than chromosome 17 where the genes for many human CC chemokines are clustered. TARC is constitutively expressed in thymus, and at lower levels in lung, colon, and small intestine. TARC is also transiently expressed in stimulated peripheral blood mononuclear cells.
Recombinant human TARC has been shown to induce chemotaxis in certain human T cell lines but not monocytes or neutrophils. TARC was recently identified to be a specific functional ligand for CCR-4, a CC chemokine receptor that is expressed in human T cell lines, peripheral T cells, basophilic cells, and cells of the megakaryocyte/platelet lineage.
TECK (thymus-expressed chemokine) is a CC chemokine that is distantly related (20-30% aa sequence identity) to other CC chemokines. Mouse TECK cDNA encodes a 142 aa residue precursor protein with a 23 aa residue signal peptide that is cleaved to yield a 119 aa residue mature protein. Human TECK cDNA has also been cloned and shown to encode a 151 aa protein that exhibits 49% aa sequence identity to mouse TECK. The expression of human and mouse TECK was shown to be highly restricted to the thymus and small intestine. Although dendritic cells has been demonstrated to be the source of TECK production in the thymus, bone-marrow-derived dendritic cells do not express TECK. The gene for mouse TECK has been mapped to chromosome 8 rather than chromosome 11 where many mouse CC chemokines are clustered. Recombinant mouse TECK has been shown to be chemotactic for activated macrophages, dendritic cells and thymocytes.37
Eotaxin-2, also named MPIF-2 and CK beta 6, is a CC chemokine recently identified in the Human Genome Sciences, Inc. database based on the presence of the CC motif and homology with other known CC chemokines.38, 39 Eotaxin-2 cDNA encodes a 119 aa residue precursor protein with a 26 aa residue signal peptide that is cleaved to generate a mature protein predicted to contain 93 aa residues with an N-glycosylation site. Although one recombinant preparation of Eotaxin-2 from insect cells was reported to be glycosylated and of the predicted size (10.5 kDa), a second preparation of recombinant Eotaxin-2 prepared from insect cells by a different group, was shown to contain a 78 aa residue carboxy-terminally truncated variant of Eotaxin-2. Additional minor carboxy-terminally truncated variants with 73, 75 and 76 residues were also isolated. Compared to other CC chemokines, Eotaxin-2 exhibits 40%, 42%, and 39% aa identity to MCP-3, MIP-1 alpha, and Eotaxin, respectively. Eotaxin-2 mRNA is weakly expressed in activated monocytes and T lymphocytes.
Recombinant Eotaxin-2 has been shown to induce chemotaxis of eosinophils, basophils, and resting T lymphocytes but not monocytes and activated T lymphocytes. Eotaxin-2 has also been shown to suppress colony formation by the high proliferative potential colony-forming cells which represent multipotential hematopoietic progenitors. On eosinophils, the effects of Eotaxin-2 were shown to be inhibited by an anti-CCR-3 antibody and to be cross-desensitized by Eotaxin or MCP-4, suggesting that all three CC chemokines act through CCR-3, at least on eosinophils.
IP-10, CRG-2 (the mouse homolog of human IP-10) and MIG are functionally related CXC chemokines that lack an ELR motif and are induced as an immediate early gene in response to IFN-gamma in macrophages and a range of cell types.40-43 Although IFN-gamma appears to be a fairly specific inducer of MIG gene expression, IP-10/CRG-2 gene expression can also be induced by IFN-alpha/beta, LPS, and viruses.
The genes for human MIG and IP-10 have been mapped to adjacent positions at chromosome 4q21, within 16 kb of one another. IP-10/CRG-2 and MIG have been shown to chemoattract activated T lymphocytes but not monocytes, neutrophils or resting T cells. Besides their roles as chemoattractants, IP-10/CRG-2 and MIG have also been shown to be inhibitors of angiogenesis and to display a potent thymus-dependent anti-tumor effect in vivo. The activities of IP-10/CRG-2 and MIG have been shown to be mediated through CXCR-3, a recently cloned chemokine receptor that is highly expressed in IL-2-activated T lymphocytes.44
Human IP-10 or mouse CRG-2 cDNA each encodes a 98 aa residue precursor protein with 21 aa residue signal peptide that is cleaved to form the 77 aa residue mature secreted protein.42, 43, 45, 46 Mature CRG-2 shares approximately 67% aa sequence identity with human IP-10. The human (mouse) MIG cDNA encodes a 125 (126) aa residue precursor protein with a 22 (21) aa residue signal peptide that is cleaved to yield a mature secreted protein predicted to contain 103 (105) aa residues.41, 47 MIG has an extended carboxy-terminus containing greater than 50% basic aa residues and is larger than most other chemokines. The carboxy-terminal residues of MIG are prone to proteolytic cleavage resulting in size heterogeneity of natural (monocyte-derived) and recombinant (CHO-derived) MIG. MIG with large carboxy-terminal deletions have been shown to have diminished activity in the calcium flux assay.
The rat chemokines CINC (cytokine-induced neutrophil chemoattractant)-1, CINC-2 alpha, CINC-2 beta, and CINC-3 (also named MIP-2) constitute a group of rat CXC chemokines that showed significant sequence similarity to human GROs and mouse MIP-2 but not IL-8.48-52 CINC-2 alpha and CINC-2 beta were originally purified as neutrophil chemoattractants from the conditioned medium of rat granulation tissue which also contain CINC-1 and CINC-3.48 Based on aa sequence analysis of the purified CINC-2 alpha protein and sequencing of the CINC-2 beta cDNA clone, both mature CINC-2 alpha and CINC-2 beta were shown to contain 68 aa residues. The aa sequences of the two CINC-2 proteins are identical except for three carboxy-terminal residues. CINC-2 beta cDNA encodes a 100 aa residue precursor protein with a 32 aa residue signal peptide that is removed to yield the mature secreted protein. At the protein sequence level, mature CINC-2 proteins are 63% identical to CINC-1 and 80% identical to CINC-3. CINC-2 proteins represent the major chemokines purified from conditioned medium of granulation tissue or LPS-induced inflammatory exudate. Other cell types known to produce CINC-2 proteins included activated macrophages and fibroblasts.
Recombinant and natural CINC-2 proteins have been shown to be specific neutrophil chemoattractants both in vivo and in vitro.53 On the basis of cross-desensitization results of various CINC proteins, it has been postulated that rat neutrophils have at least two classes of CINC receptors: 1) CINC-3-specific receptors; and 2) a common receptor shared by all CINCs.54, 55
Human MCP-4 and mouse MCP-5 are recent additions to the list of monocyte chemoattractant proteins, a group of CC chemokines.
MCP-4 cDNA encodes a 98 aa residue precursor protein with a 23 aa residue hydrophobic signal peptide that is cleaved to yield a 8 kDa, 75 aa mature MCP-4. Mature MCP-4 lacks any potential N-glycosylation sites and shares a pyroglutamate proline motif with other human MCP proteins. Human MCP-4 is most homologous to MCP-1, MCP-3 and Eotaxin, exhibiting approximately 65-66% aa sequence identity.56 MCP-4 mRNA is expressed by a number of activated cell types, including endothelial cells,56, 57 macrophages,58 bronchial epithelium and type II alveolar cells,1 and perhaps lymphocytes.56 MCP-4 is a chemoattractant for monocytes and eosinophils, and activates basophils.56, 57, 59, 60 In addition, it has been reported to be chemotactic for CD4+ and CD8+ T cells, with an activity almost equivalent to that of MCP-3.59 The bioactivities of MCP-4 are most likely mediated by the CC chemokine receptors CCR-261 and CCR-3,62, 63 both of which have been shown to bind MCP-4.56-58, 60 Although many factors may be involved, the fact that the respective Th1 and Th2 cytokines, IFN-gamma and IL-4, consistently synergize with other cytokines to upregulate MCP-4 expression may help explain why monocytes, eosinophils and basophils are often found at the sites of type 1 and type 2 immune responses.56
Mouse MCP-5 cDNA encodes a 104 aa residue precursor protein with a 22 aa residue hydrophobic signal peptide that is cleaved to yield a 82 aa residue mature protein. Mouse MCP-5 is most homologous to human MCP-1 (66% aa sequence identity). Mouse MCP-5 has been shown to be a strong chemoattractant towards monocytes, but only modest activity towards eosinophils.64 At this time, it is known that MCP-5 is expressed by macrophages (and possibly neutrophils); is species cross-reactive; and utilizes CCR-2 as a receptor, although there is likely to be another MCP-5 receptor yet to be discovered.64
Macrophage-derived chemokine (MDC)65 also named stimulated T cell chemotactic protein (STCP-1)66 is a newly-discovered CC chemokine initially isolated from clones of monocyte-derived macrophages. Human MDC cDNA encodes a precursor protein of 93 aa residues with a 24 aa residue signal peptide that is cleaved to yield the 69 aa residue mature 8kDa protein. At the aa sequence level, MDC shows less than 35% identity to other CC chemokine members. Human MDC is expressed in dendritic cells (DC), macrophages and activated monocytes.65, 66 In addition, MDC expression is also detected in the tissues of thymus, lymph node and appendix. Recombinant or chemically synthesized mature synthesized MDC has been shown to induce chemotaxis or Ca2+ mobilization in dendritic cells, IL-2 activated NK cells, and activated T lymphocytes.65, 66 Functionally, MDC may well be part of a recruitment cascade where DC are directed into the site of initiation of an immune response via the macrophage-derived MDC, naive T cells are induced to follow via another newly-discovered DC chemokine DC-CK1.67 Recently, MDC has been shown to suppress infection of CD8+ cell-depleted peripheral blood mononuclear cells by primary non-syncytium-inducing and syncytium-inducing isolated of HIV-1 and T cell line-adapted isolate HIV-1.68 The receptor(s) for MDC is currently unknown.