First printed in R&D Systems' 1997 Catalog.
|Figure 1. M-trophic HIV-1 & chemokine receptors. 1. gp120 and gp41 are formed intracellularly by the cleavage of a 160 kDa precursor; The two subunits heterodimerize non-covalently. 2. CD4 binds to gp120 binding sites distributed along the C-terminal region of gp120. 3. Following binding to CD4, plus a change in gp120 conformation, V3 region binds to CKR5. 4. gp120 “dissociates” from gp41, forming soluble gp120. 5. Upon exposure of gp41 to the macrophage cell membrane, allowing passage of viral nucleic acid into macrophage cytoplasm (arrow).
Chemokines (alternative names are intercrines, PF-4 superfamily of cytokines or SIS cytokines) constitute a superfamily of small (8-10 kDa), inducible, secreted, pro-inflammatory cytokines that are involved in a variety of immune and inflammatory responses, acting primarily as chemoattractants and activators of specific types of leukocytes (for recent reviews, see references 1-10). Some members of this family were initially identified on the basis of their biological activities (e.g., IL-8, GRO), others were discovered using subtractive hybridization (e.g., RANTES) or signal sequence trap (e.g., PBSF/SDF-1)11 cloning strategies. Members of the chemokine family exhibit from 20% to over 90% identity in their predicted amino acid sequences.
Three classes of chemokines have been defined by the arrangement of the conserved cysteine (C) residues of the mature proteins: the CXC or a chemokines that have one amino acid residue separating the first two conserved cysteine residues; the CC or beta chemokines in which the first two conserved cysteine residues are adjacent; the C or gamma chemokines which lack two (the first and third) of the four conserved cysteine residues. Within the CXC subfamily, the chemokines can be further divided into two groups. One group of the CXC chemokines have the characteristic three amino acid sequence ELR (glutamic acid-leucine-arginine) motif immediately preceding the first cysteine residue near the amino terminus. A second group of CXC chemokines lack such an ELR domain. The CXC chemokines with the ELR domain (including IL-8, GRO alpha/beta/gamma, mouse KC, mouse MIP-2, ENA-78, GCP-2, PBP/CTAPIII/beta-TG/NAP-2) act primarily on neutrophils as chemoattractants and activators, inducing neutrophil degranulation with release of myeloperoxidase and other enzymes. The CXC chemokines without the ELR domain (e.g., IP-10/mouse CRG, Mig, PBSF/SDF-1, PF4), the CC chemokines (e.g., MIP-1 alpha, MIP-1 beta, RANTES, MCP-1/2/3/4/mouse JE/mouse MARC, eotaxin, I-309/TCA3, HCC-1, C10), and the C chemokines (e.g., lymphotactin), chemoattract and activate monocytes, dendritic cells, T-lymphocytes, natural killer cells, B-lymphocytes, basophils, and eosinophils.
In addition to their roles in regulating leukocyte recruitment and trafficking, certain chemokines have been reported to act on hematopoietic progenitor cells and on non-leukocytic cells such as fibroblasts, smooth muscle cells, keratinocytes and melanoma cell lines. Other chemokines have also been implicated as playing a role in wound healing, in angiogenesis and in viral infection. In in vitro assays, chemokines have overlapping and redundant functions. It remains to be determined to what extent the various chemokines have unique roles in vivo. To date, over 20 chemokines have been cloned and characterized. Additional chemokines have also turned up in various cloning strategies. The genes for all CC chemokines have been found to cluster on human chromosome 17q and mouse chromosome 11. With the exception of PBSF/SDF-1, all CXC chemokines genes have been found to cluster on human chromosome 4q. The human PBSF/SDF-1 gene12 and the gene for the C chemokine lymphotactin13 have been localized to human chromosome 10q and 1, respectively. The clustering of chemokine genes suggests that many cytokine family members arose through gene duplication and subsequent divergence.
Chemokines bind to heparin and glycosaminoglycans on cell surface proteoglycans.14, 15 The immobilization of chemokines by cell surface proteoglycans or components of the extracellular matrix is thought to be important for the maintenance of the chemokine gradient needed for leukocyte activation and diapedesis and migration into tissue spaces.
|Figure 2. Chemokine receptors.
Chemokines mediate their activities by binding to target cell surface chemokine receptors that belong to the large family of G protein-coupled, seven transmembrane (7 TM) domain receptors (also called serpentine receptors). Based on the receptor nomenclature established at the 1996 Gordon Research Conference on chemotactic cytokines, the chemokine receptors that bind CXC chemokines are designated CXCRs and the receptors that bind CC chemokines are designated CCRs. To date, four CXC chemokine receptors (CXCR-1 through 4) and five CC chemokine receptors (CCR-1 through 5) have been cloned and characterized.15, 16 In addition, the Duffy blood group antigen (DARC) has been shown to be an erythrocyte chemokine receptor that can bind selected CXC, as well as CC chemokines.17 Two virally encoded chemokine receptors, a CC receptor encoded by a cytomegalovirus open reading frame CMV US28,18 and a CXC receptor encoded by herpes saimiri virus open reading frame, HSV ECRF3,19 have also been described. Leukocytes have generally been found to express more than one receptor type. The various CXCRs and CCRs are known to exhibit overlapping ligand specificities.
CXCR-1 and CXCR-2, previously known as IL-8RA, or type I IL-8 receptor, and IL-8RB, or type II IL-8 receptor, respectively, have been shown to share approximately 77% amino acid sequence identity. IL-8 binds to both receptors with high affinity and induces rapid elevation of cytosolic Ca++ levels.20-22 Whereas CXCR-1 is highly specific for IL-8, CXCR-2 has broad specificity and has been shown to bind with high-affinity to other ELR motif containing a chemokines including GRO alpha, GRO beta, GRO gamma, NAP-2 and ENA-78. In contrast, PF4 and IP-10, two a chemokines that lack the ELR motif, have been shown to lack binding affinity for CXCR-2. CXCR-1 and CXCR-2 are expressed by neutrophils but not B lymphocytes and T lymphocytes. CXCR-3, also known as the IP-10/Mig receptor, is a recently cloned chemokine receptor that shares approximately 40% protein sequence identity with CXCR-1 and CXCR-2, and 34.2 - 36.9% amino acid sequence identity with the five known CC chemokine receptors.23 CXCR-3 is highly expressed by IL-2- activated T lymphocytes but not by resting T lymphocytes, B lymphocytes, monocytes and granulocytes. CXCR-3 binds IP-10 and Mig, but not PF4, with high affinity and mediates Ca++ mobilization and chemotaxis. CXCR-3 does not bind any of the CXC chemokines containing the ELR motif. CXCR-4, also known as fusin or LESTR,24, 25 was originally discovered as an orphan receptor with structural similarity to chemokine receptors. CXCR-4 was subsequently identified as a necessary cofactor for entry of T cell-tropic HIV viruses into CD4+ cells.24 The CXC chemokine PBSF/SDF-1 has now been shown to be the ligand for CXCR-4 and a powerful inhibitor of infection by T cell-tropic HIV-1 strains.26, 27
CCR-1 (MIP-1 alpha/RANTES receptor) was the first identified C-C chemokine receptor and is expressed on monocytes, neutrophils or eosinophils. CCR-1 binds MIP-1 alpha, RANTES, MCP-3 with high affinity and also MIP-1 beta, and MCP-1 with lower affinity.28, 29 CCR-2A and CCR-2B (MCP-1RA and MCP-1RB) differ in their alternatively spliced carboxy-terminus and are probably spliced variants of a single gene.30 CCR-2A and B specifically bind MCP-1 and MCP-3. The two receptors are expressed on monocytes but not on neutrophils or eosinophils. CCR-3 is a high affinity receptor for eotaxin, an eosinophil-specific chemo-attractant.31 -34 In humans, CCR-3 was found to be expressed exclusively on eosinophils. CCR-4 was originally cloned from a human immature basophilic cell line and has since been shown to be expressed in T cells and IL-5-primed basophils. CCR-4 has been shown to mediate the biological activities of RANTES, MIP-1 alpha and MCP-1. CCR-5 is the most recently discovered CC receptor and has 48 - 75% amino acid sequence identity to the other CC receptors.35, 36 CCR-5 is expressed in primary adherent monocytes, but not in neutrophils or eosinophils.34, 37-40 CCR-5 mediates the activities of MIP-1 alpha, MIP-1 beta and RANTES. Recently CCR-5 has also been shown to be a co-receptor on CD4+ target cells for infection with primary, monocyte-tropic HIV-1 viruses.41-43
Erythrocyte Chemokine Receptor and Virally Encoded Chemokine Receptors
A multispecific chemokine receptor that binds many a chemokines (IL-8, GRO, NAP-2) as well as beta chemokines (MCP-1, RANTES, but not MIP-1 alpha or MIP-1 beta) with high affinity has been identified on erythrocyte surfaces.17, 44, 45 This receptor has now been shown to be identical to the DARC which is known to be the receptor for invasion of the malarial parasite Plasmodium vivax. Recently, an isoform of this receptor has also been localized to the postcapillary venule endothelial cells in kidney.46 At present, the physiological role of this receptor is unknown. It has been speculated that this receptor may have a role in maintaining a low plasma IL-8 concentration so that circulating neutrophils do not become desensitized and thus unresponsive to changes in tissue IL-8 concentrations.
Viral open reading frames (ORFs) that encode chemokine receptor homologues have been reported.18,19 The ORF US28 of the human Cytomegalovirus encodes a protein that exhibits approximately 30% sequence identity with CCR-1. When transfected into 293 fibroblasts, this receptor has been reported to bind MIP-1 beta, MIP-1 beta, MCP-1 and RANTES, but not a chemokines. Similarly, Herpes virus saimiri ORF ECRF3 also encodes a protein that shares 30% sequence identity with the IL-8 receptors. When transfected into frog oocytes, this receptor can transduce a signal in response to IL-8, GRO alpha, and NAP-2. The role of the viral chemokines receptors in the pathogenesis of virus infections remains to be determined.
Chemokines and HIV-1 Infection
During the past year, the research areas dealing with the mechanisms involved in HIV infection and with the mediation of biological effects by chemokines have become closely intertwined.
Human type I immunodeficiency viruses (HIV-1) infect CD4+ target cells.47, 48 The entry of HIV-1 into target cells requires the binding of the viral envelope glycoprotein (Env) with the target cell CD4 and an additional target cell co-receptor.49 Primary HIV-1 isolates from patients have been found to infect monocyte/macrophages and primary T cells, but not CD4+ transformed cell lines. These so-called macrophage-tropic HIV-1 are typically nonsyncytium-inducing (NSI) and predominate during the asymptomatic early stages of HIV infection. Other HIV-1 isolates, referred to as T cell-tropic virus, infect CD4+ cell lines and primary T cells, but not macrophages. These T cell-tropic viruses are syncytium-inducing (SI) and appear late in AIDS development.50 The difference in HIV cell tropism has been attributed to specific sequence differences in the V3 loop of the gp120 subunit of the envelope proteins of the different HIV isolates.51
The co-receptors required for the fusion of the T cell-tropic and macrophage-tropic viruses with their target cells have now been identified to be fusin and CCR-5, respectively.24, 25 Fusin is a 7 TM domain protein with significant amino acid sequence homology to the IL-8 receptors. A CXC chemokine, PBSF/SDF-1, has recently been identified to be a ligand for fusin.26, 27 The identification of CCR-5 as the co-receptor for macrophage-tropic viruses is consistent with an earlier report identifying the CCR-5 ligands (RANTES, MIP-1 alpha and MIP-1 beta) as the major HIV suppressive factors produced by CD8+ T cells for macrophage-tropic, but not T cell tropic, HIV isolates.52 Besides fusin and CCR-5, use of other chemokine receptors such as CCR-2B and CCR-3 by a minority of HIV isolates has also been reported.53
Whereas the vast majority of the population are susceptible to HIV infection, a small number of individuals that have been exposed repeatedly to HIV remained uninfected. Studies on two of these exposed/uninfected (EU) individuals have revealed a homozygous defect in their CCR-5 HIV co-receptor, apparently accounting for their resistance to macrophage-tropic virus. The affected individuals expressed a truncated CCR-5 that was not detected on cell surfaces but otherwise displayed no other phenotype. These EU individuals have been shown to have unaltered fusin. Several other chemokine receptors were also shown to be unmodified. The CD4+ T cells from these individuals have been shown to be readily infected by T cell-tropic viruses. Based on these findings, it would appear that macrophage-tropic isolates are likely to be the viruses responsible for the transmission of HIV-1 viruses and that CCR-5 may play a critical role in the transmission and progression of AIDS.54, 55
Human HCC-1 (for hemofiltrate CC chemokine 1) is a newly discovered, 8.7 kDa nonglycosylated polypeptide originally isolated from the hemofiltrate of chronic renal failure patients.56 The mature molecule is 74 amino acids in length, and shows 46% overall aa identity with the CC chemokines MIP-1 alpha and MIP-1 beta. Between the first and last cysteine residue of these proteins, amino acid identity is as high as 61%. Like MIP-1 alpha, HCC-1 induces the proliferation of CD34+ myeloid progenitor cells (albeit at significantly increased concentrations). Unlike MIP-1 alpha, HCC-1 shows weak chemotactic activity towards monocytes.56 As a chemokine, HCC-1 is very unusual in that it is found in normal plasma at significant concentrations. Concentrations are reported to range from 1.5-10.0 nM in normal plasma to 2.5-80.0 nM in blood from patients with chronic renal failure. The natural source(s) for HCC-1 is unknown. To date, no receptor for HCC-1 has been identified. However, it appears to recognize at least one receptor for MIP-1 alpha, extending the possibilities to CCR-1, 4 and 5.16 HCC-1 was also identified in the HGS/TGIR database and named macrophage colony inhibition factor (M-CIF). It has been shown to be a potent inhibitor of colony formation in response to M-CSF.61
SDF-1 alpha and SDF-1 beta (for stromal cell derived factor 1 alpha and beta) are the first cytokines identified using the signal sequence trap cloning strategy from a mouse bone-marrow stromal cell line.11 These proteins were subsequently also cloned from a human stromal cell line as cytokines that supported the proliferation of a stromal cell-dependent pre-B-cell line and named pre-B cell growth stimulating factor (PBSF).57 SDF-1 alpha and SDF-1 beta cDNAs encode precursor secreted proteins of 89 and 93 amino acid residues, respectively. Both SDF-1 alpha and SDF-1 beta are encoded by a single gene and arise by alternative splicing.12 The two proteins are identical except for the four amino acid residues that are present in the carboxy-terminus of SDF-1 beta and absent from SDF-1 beta. Based on their amino acid sequence characteristics, SDF-1 alpha and SDF-1 beta are classified in the CXC subfamily of chemokines. Unlike other known chemokine alpha and beta subfamily members that cluster on chromosomes 4 and 17, respectively, the gene for SDF-1 has now been localized to chromosome 10q11.1.12 The nucleotide and amino acid sequences of SDF-1 alpha and SDF-1 beta are highly conserved between species, showing only one amino acid substitution between the human and mouse proteins. SDF-1 amino acid sequence also shows equal homology to both CC and CXC subfamily chemokines. SDF-1, unlike other chemokine family members, has been found to be constitutively expressed in a broad range of tissues.11, 12, 57, 59
These distinctions of SDF-1 from other CC and CXC chemokines suggest that SDF-1 may have unique functions different from those of other chemokine family members. PBSF/SDF-1 is a pre-B cell stimulatory factor as well as a potent chemoattractant for T-lymphocytes and monocytes, but not neutrophils. PBSF/SDF-1 knock-out mice lacking the SDF-1 proteins show reduced B-cell progenitors in fetal liver and bone marrow. However, myeloid progenitors in these mice were reduced only in the bone marrow, but not in the fetal liver.60 Recently, PBSF/SDF-1 was shown to be a ligand for CXCR-4, the chemokine receptor that functions as a co-receptor for lymphocyte-tropic HIV-1 strains.24 PBSF/SDF-1 has also been found to be a powerful inhibitor of infection by lymphocyte-tropic HIV-1 virus strains.26, 27
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