CCL5/CCR5生物学轴在肿瘤中作用的研究进展

肿瘤因其易转移、易复发的特性成为一大难以治愈的疾病,已严重威胁到人类的生命健康。肿瘤微环境在肿瘤的生长、迁移、免疫逃逸、血管生成等过程中具有明显的促进作用。肿瘤微环境中细胞分泌的CCL5发挥的作用已受到越来越多的关注,且许多研究表明抑制CCL5/CCR5生物学轴可抑制肿瘤迁移、血管生成等,预示着这可能成为一个新的肿瘤治疗策略。本文总结了近年来关于CCL5/CCR5生物学轴的研究,包括CCL5/CCR5生物学轴介导的肿瘤生长迁移、血管生成、免疫逃逸等作用,及CCR5抑制剂在肿瘤治疗中的广阔前景。     

CCR5 deficiency aggravates crescentic glomerulonephritis in mice

The chemokine receptor CCR5 is predominantly expressed on monocytes and Th1-polarized T cells, and plays an important role in T cell and monocyte recruitment in inflammatory diseases. To investigate the functional role of CCR5 in renal inflammation, we induced a T cell-dependent model of glomerulonephritis (nephrotoxic serum nephritis) in CCR5(-/-) mice. Induction of nephritis in wild-type mice resulted in up-regulation of renal mRNA expression of the three CCR5 chemokine ligands, CCL5 (15-fold), CCL3 (4.9-fold), and CCL4 (3.4-fold), in the autologous phase of the disease at day 10. The up-regulated chemokine expression was paralleled by infiltration of monocytes and T cells, followed by renal tissue injury, albuminuria, and loss of renal function. Nephritic CCR5(-/-) mice showed a 3- to 4-fold increased renal expression of CCL5 (61.6-fold vs controls) and CCL3 (14.1-fold vs controls), but not of CCL4, in comparison with nephritic wild-type mice, which was accompanied by augmented renal T cell and monocyte recruitment and increased lethality due to uremia. Furthermore, CCR5(-/-) mice showed an increased renal Th1 response, whereas their systemic humoral and cellular immune responses were unaltered. Because the CCR5 ligands CCL5 and CCL3 also act via CCR1, we investigated the effects of the pharmacological CCR1 antagonist BX471. CCR1 blockade in CCR5(-/-) mice significantly reduced renal chemokine expression, T cell infiltration, and glomerular crescent formation, indicating that increased renal leukocyte recruitment and consecutive tissue damage in nephritic CCR5(-/-) mice depended on functional CCR1. In conclusion, this study shows that CCR5 deficiency aggravates glomerulonephritis via enhanced CCL3/CCL5-CCR1-driven renal T cell recruitment.     

Importance of the CCR5-CCL5 axis for mucosal Trypanosoma cruzi protection and B cell activation

Trypanosoma cruzi is an intracellular parasite and the causative agent of Chagas disease. Previous work has shown that the chemokine receptor CCR5 plays a role in systemic T. cruzi protection. We evaluated the importance of CCR5 and CCL5 for mucosal protection against natural oral and conjunctival T. cruzi challenges. T. cruzi-immune CCR5(-/-) and wild-type C57BL/6 mice were generated by repeated infectious challenges with T. cruzi. CCR5(-/-) and wild-type mice developed equivalent levels of cellular, humoral, and protective mucosal responses. However, CCR5(-/-)-immune mice produced increased levels of CCL5 in protected gastric tissues, suggesting compensatory signaling through additional receptors. Neutralization of CCL5 in CCR5(-/-)-immune mice resulted in decreased mucosal inflammatory responses, reduced T. cruzi-specific Ab-secreting cells, and significantly less mucosal T. cruzi protection, confirming an important role for CCL5 in optimal immune control of T. cruzi replication at the point of initial mucosal invasion. To investigate further the mechanism responsible for mucosal protection mediated by CCL5-CCR5 signaling, we evaluated the effects of CCL5 on B cells. CCL5 enhanced proliferation and IgM secretion in highly purified B cells triggered by suboptimal doses of LPS. In addition, neutralization of endogenous CCL5 inhibited B cell proliferation and IgM secretion during stimulation of highly purified B cells, indicating that B cell production of CCL5 has important autocrine effects. These findings demonstrate direct effects of CCL5 on B cells, with significant implications for the development of mucosal adjuvants, and further suggest that CCL5 may be important as a general B cell coactivator.     

New insights into the mechanisms whereby low molecular weight CCR5 ligands inhibit HIV-1 infection

CC chemokine receptor 5 (CCR5) is a G-protein-coupled receptor for the chemokines CCL3, -4, and -5 and a coreceptor for entry of R5-tropic strains of human immunodeficiency virus type 1 (HIV-1) into CD4(+) T-cells. We investigated the mechanisms whereby nonpeptidic, low molecular weight CCR5 ligands block HIV-1 entry and infection. Displacement binding assays and dissociation kinetics demonstrated that two of these molecules, i.e. TAK779 and maraviroc (MVC), inhibit CCL3 and the HIV-1 envelope glycoprotein gp120 binding to CCR5 by a noncompetitive and allosteric mechanism, supporting the view that they bind to regions of CCR5 distinct from the gp120- and CCL3-binding sites. We observed that TAK779 and MVC are full and weak inverse agonists for CCR5, respectively, indicating that they stabilize distinct CCR5 conformations with impaired abilities to activate G-proteins. Dissociation of [(125)I]CCL3 from CCR5 was accelerated by TAK779, to a lesser extent by MVC, and by GTP analogs, suggesting that inverse agonism contributes to allosteric inhibition of the chemokine binding to CCR5. TAK779 and MVC also promote dissociation of [(35)S]gp120 from CCR5 with an efficiency that correlates with their ability to act as inverse agonists. Displacement experiments revealed that affinities of MVC and TAK779 for the [(35)S]gp120-binding receptors are in the same range (IC(50) ～6.4 versus 22 nm), although we found that MVC is 100-fold more potent than TAK779 for inhibiting HIV infection. This suggests that allosteric CCR5 inhibitors not only act by blocking gp120 binding but also alter distinct steps of CCR5 usage in the course of HIV infection.     

Assignment of immune-related genes to the channel catfish, Ictalurus punctatus, genetic map

Eighteen new genes, adenosine A1 receptor (ADORA1), complement component 4-beta (C4b), complement component 8-beta (C8b), chemokine ligand 19 (CCL19), chemokine ligand 21 (CCL21), chemokine ligand 25 (CCL25), chemokine receptor 2 (CCR2), chemokine receptor 5 (CCR5), chemokine receptor 4 (CCR4), chemokine receptor 7 (CCR7), chemokine receptor 9 (CCR9), interleukin 1-beta (IL1B), integrin II-beta (ITGB2), novel immune type receptor 2 (NITR2), novel immune type receptor 4 (NITR4), natural killer cell lysin (NKLYSIN), nucleotide excision repair (RAD23B) and tumour necrosis factor-alpha (TNF), were assigned to the channel catfish (Ictalurus punctatus) genetic linkage map. Polymorphic microsatellite markers were developed for NITR2, NITR4 and RAD23B from short-tandem repeats in the available sequence. Polymorphic microsatellite markers were developed for the remaining 15 genes by short-tandem repeat-anchored primer sequencing of catfish bacterial artificial chromosomes. Two gene clusters (MYOG-NRAMP-ADORA1) and (CCR4-CCR2-CCR5) displayed conservation of synteny between catfish and mammals. Assignment of 18 new genes to the catfish linkage map will further advance integration of genetic and physical maps and comparative mapping between channel catfish and map rich species.     

A competitive binding study of chemokine, sulfated receptor, and glycosaminoglycan interactions by nano-electrospray ionization mass spectrometry

Chemokines are secreted proteins that play roles in inducing chemotaxis, extravasation, and activation of leukocytes associated with inflammatory or homeostatic processes. Tyrosine sulfation of the chemokine receptor has been shown to be important for binding and signaling. We have applied a mass spectrometry method to measure the contribution of this posttranslational modification to binding of its ligand chemokine. Using nano-electrospray time-of-flight mass spectrometry (nano-ESI TOF MS), we determined the association constants of C-C motif chemokine 7 (CCL7) with C-C chemokine receptor type 2 (CCR2), monosulfated CCR2, and disulfated CCR2 peptides to be 1.1 × 10⁴ M⁻¹, 3.9 × 10⁴ M⁻¹, and 4.0 × 10⁵ M⁻¹, respectively. To our knowledge, this is the first reported association constant measurement between a protein and sulfated peptide using MS. Furthermore, nano-ESI MS was used to characterize noncovalent binding interactions among CCL7, Arixtra (a pentasaccharide glycosaminoglycan [GAG] analog), and disulfated CCR2 peptide. A lack of observable ternary complex formation prompted investigation of competitive binding. Results of this study suggest that CCR2 competes partially with GAG for CCL7 binding and that disulfated CCR2 peptide has a higher binding affinity than Arixtra, which correlates with data from association constant measurements for CCL7-disulfated CCR2 and CCL7-Arixtra.     

Angiotensin-converting Enzyme Inhibition Down-regulates the Pro-atherogenic Chemokine Receptor 9 (CCR9)-Chemokine Ligand 25 (CCL25) Axis

Many experimental and clinical studies suggest a relationship between enhanced angiotensin II release by the angiotensin-converting enzyme (ACE) and the pathophysiology of atherosclerosis. The atherosclerosis-enhancing effects of angiotensin II are complex and incompletely understood. To identify anti-atherogenic target genes, we performed microarray gene expression profiling of the aorta during atherosclerosis prevention with the ACE inhibitor, captopril. Atherosclerosis-prone apolipoprotein E (apoE)-deficient mice were used as a model to decipher susceptible genes regulated during atherosclerosis prevention with captopril. Microarray gene expression profiling and immunohistology revealed that captopril treatment for 7 months strongly decreased the recruitment of pro-atherogenic immune cells into the aorta. Captopril-mediated inhibition of plaque-infiltrating immune cells involved down-regulation of the C-C chemokine receptor 9 (CCR9). Reduced cell migration correlated with decreased numbers of aorta-resident cells expressing the CCR9-specific chemoattractant factor, chemokine ligand 25 (CCL25). The CCL25-CCR9 axis was pro-atherogenic, because inhibition of CCR9 by RNA interference in hematopoietic progenitors of apoE-deficient mice significantly retarded the development of atherosclerosis. Analysis of coronary artery biopsy specimens of patients with coronary artery atherosclerosis undergoing bypass surgery also showed strong infiltrates of CCR9-positive cells in atherosclerotic lesions. Thus, the C-C chemokine receptor, CCR9, exerts a significant role in atherosclerosis.     

CCR6, the Sole Receptor for the Chemokine CCL20, Promotes Spontaneous Intestinal Tumorigenesis

Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been associated with colorectal cancer growth and metastasis, however, a causal role for CCL20 signaling through CCR6 in promoting intestinal carcinogenesis has not been demonstrated in vivo. In this study, we aimed to determine the role of CCL20-CCR6 interactions in spontaneous intestinal tumorigenesis. CCR6-deficient mice were crossed with mice heterozygous for a mutation in the adenomatous polyposis coli (APC) gene (APC^MIN/+ mice) to generate APC^MIN/+ mice with CCR6 knocked out (CCR6KO-APC^MIN/+ mice). CCR6KO-APC^MIN/+ mice had diminished spontaneous intestinal tumorigenesis. CCR6KO-APC^MIN/+ also had normal sized spleens as compared to the enlarged spleens found in APC^MIN/+ mice. Decreased macrophage infiltration into intestinal adenomas and non-tumor epithelium was observed in CCR6KO-APC^MIN/+ as compared to APC^MIN/+ mice. CCL20 signaling through CCR6 caused increased production of CCL20 by colorectal cancer cell lines. Furthermore, CCL20 had a direct mitogenic effect on colorectal cancer cells. Thus, interactions between CCL20 and CCR6 promote intestinal carcinogenesis. Our results suggest that the intestinal tumorigenesis driven by CCL20-CCR6 interactions may be driven by macrophage recruitment into the intestine as well as proliferation of neoplastic epithelial cells. This interaction could be targeted for the treatment or prevention of malignancy.     

Preferential migration of regulatory T cells mediated by gliomasecreted chemokines can be blocked with chemotherapy

Despite the immunogenicity of glioblastoma multiforme (GBM), immune-mediated eradication of these tumors remains deficient. Regulatory T cells (Tregs) in the blood and within the tumor microenvironment of GBM patients are known to contribute to their dismal immune responses. Here, we determined which chemokine secreted by gliomas can preferentially induce Treg recruitment and migration. In the malignant human glioma cell lines D-54, U-87, U-251, and LN-229, the chemokines CCL22 and CCL2 were detected by intracellular cytokine analysis. Furthermore, tumor cells from eight patients with GBM had a similar chemokine expression profile. However, only CCL2 was detected by enzyme-linked immunosorbent assay, indicating that CCL2 may be the principal chemokine for Treg migration in GBM patients. Interestingly, the Tregs from GBM patients had significantly higher expression levels of the CCL2 receptor CCR4 than did Tregs from healthy controls. Glioma supernatants and the recombinant human chemokines CCL2 and CCL22 induced Treg migration and were blocked by antibodies to the chemokine receptors. Production of CCL2 by glioma cells could also be mitigated by the chemotherapeutic agents temozolomide and carmustine [3-bis (2-chloroethyl)-1-nitrosourea]. Our results indicate that gliomas augment immunosuppression by selective chemokine-mediated recruitment of Tregs into the tumor microenvironment and that modulating this interaction with chemotherapy could facilitate the development of novel immunotherapeutics to malignant gliomas. Justin T. Jordan and Wei Sun are contributed equally to this work. An erratum to this article can be found at     

Expression and purification of recombinant human CCL5 and its biological characterization

The Protein Journal - C-C motif chemokine ligand 5 (CCL5) is crucial in the tumor microenvironment. It has been previously reported to act as a key role in tumor invasion and metastasis. However,...     

CCL3-CCR5 axis regulates intratumoral accumulation of leukocytes and fibroblasts and promotes angiogenesis in murine lung metastasis process

Metastasis proceeds through interaction between cancer cells and resident cells such as leukocytes and fibroblasts. An i.v. injection of a mouse renal cell carcinoma, Renca, into wild-type mice resulted in multiple metastasis foci in lungs and was associated with intratumoral accumulation of macrophages, granulocytes, and fibroblasts. A chemokine, CCL3, was detected in infiltrating cells and, to a lesser degree, tumor cells, together with an infiltration of leukocytes expressing CCR5, a specific receptor for CCL3. A deficiency of the CCL3 or CCR5 gene markedly reduced the number of metastasis foci in the lung, and the analysis using bone marrow chimeric mice revealed that both bone marrow- and non-bone marrow-derived cells contributed to metastasis formation. CCL3- and CCR5-deficient mice exhibited a reduction in intratumoral accumulation of macrophages, granulocytes, and fibroblasts. Moreover, intratumoral neovascularization, an indispensable process for metastasis, was attenuated in these gene-deficient mice. Intrapulmonary expression of matrix metalloproteinase (MMP)-9 and hepatocyte growth factor (HGF) was enhanced in wild-type mice, and the increases were markedly diminished in CCL3- and CCR5-deficient mice. Furthermore, MMP-9 protein was detected in macrophages and granulocytes, the cells that also express CCR5 and in vitro stimulation by CCL3-induced macrophages to express MMP-9. Intratumoral fibroblasts expressed CCR5 and HGF protein. In vitro CCL3 stimulated fibroblasts to express HGF. Collectively, the CCL3-CCR5 axis appears to regulate intratumoral trafficking of leukocytes and fibroblasts, as well as MMP-9 and HGF expression, and as a consequence to accelerate neovascularization and subsequent metastasis formation.     

Role of CCL3L1-CCR5 genotypes in the epidemic spread of HIV-1 and evaluation of vaccine efficacy

Background

Polymorphisms in CCR5, the major coreceptor for HIV, and CCL3L1, a potent CCR5 ligand and HIV-suppressive chemokine, are determinants of HIV-AIDS susceptibility. Here, we mathematically modeled the potential impact of these genetic factors on the epidemic spread of HIV, as well as on its prevention.

Methods and Results

Ro, the basic reproductive number, is a fundamental concept in explaining the emergence and persistence of epidemics. By modeling sexual transmission among HIV+/HIV− partner pairs, we find that Ro estimates, and concordantly, the temporal and spatial patterns of HIV outgrowth are highly dependent on the infecting partners'' CCL3L1-CCR5 genotype. Ro was least and highest when the infected partner possessed protective and detrimental CCL3L1-CCR5 genotypes, respectively. The modeling data indicate that in populations such as Pygmies with a high CCL3L1 gene dose and protective CCR5 genotypes, the spread of HIV might be minimal. Additionally, Pc, the critical vaccination proportion, an estimate of the fraction of the population that must be vaccinated successfully to eradicate an epidemic was <1 only when the infected partner had a protective CCL3L1-CCR5 genotype. Since in practice Pc cannot be >1, to prevent epidemic spread, population groups defined by specific CCL3L1-CCR5 genotypes might require repeated vaccination, or as our models suggest, a vaccine with an efficacy of >70%. Further, failure to account for CCL3L1-CCR5-based genetic risk might confound estimates of vaccine efficacy. For example, in a modeled trial of 500 subjects, misallocation of CCL3L1-CCR5 genotype of only 25 (5%) subjects between placebo and vaccine arms results in a relative error of ∼12% from the true vaccine efficacy.

Conclusions

CCL3L1-CCR5 genotypes may impact on the dynamics of the HIV epidemic and, consequently, the observed heterogeneous global distribution of HIV infection. As Ro is lowest when the infecting partner has beneficial CCL3L1-CCR5 genotypes, we infer that therapeutic vaccines directed towards reducing the infectivity of the host may play a role in halting epidemic spread. Further, CCL3L1-CCR5 genotype may provide critical guidance for optimizing the design and evaluation of HIV-1 vaccine trials and prevention programs.     

CCL27-CCR10 interactions regulate T cell-mediated skin inflammation

The skin-associated chemokine CCL27 (also called CTACK, ALP and ESkine) and its receptor CCR10 (GPR-2) mediate chemotactic responses of skin-homing T cells in vitro. Here we report that most skin-infiltrating lymphocytes in patients suffering from psoriasis, atopic or allergic-contact dermatitis express CCR10. Epidermal basal keratinocytes produced CCL27 protein that bound to extracellular matrix, mediated adhesion and was displayed on the surface of dermal endothelial cells. Tumor necrosis factor-alpha and interleukin-1beta induced CCL27 production whereas the glucocorticosteroid clobetasol propionate suppressed it. Circulating skin-homing CLA+ T cells, dermal microvascular endothelial cells and fibroblasts expressed CCR10 on their cell surface. In vivo, intracutaneous CCL27 injection attracted lymphocytes and, conversely, neutralization of CCL27-CCR10 interactions impaired lymphocyte recruitment to the skin leading to the suppression of allergen-induced skin inflammation. Together, these findings indicate that CCL27-CCR10 interactions have a pivotal role in T cell-mediated skin inflammation.     

CCL17-CCR4 axis promotes metastasis via ERK/MMP13 pathway in bladder cancer

As an important chemokine receptor, the role of CCR4 in the progression of bladder cancer (BC) remains unknown. In this study, we have shown that CCR4 expression was upregulated in bladder carcinoma tissues compared with adjacent nontumor tissues. Kaplan-Meier survival analysis revealed that CCR4 expression was an independent prognostic risk factor in BC patients, and the addition of CCL17 induced CCR4 production and promoted migration and invasion of BC cells. In addition, CCR4 knockdown significantly attenuated the migratory and invasive capabilities of BC cells. Mechanistically, CCL17-CCR4 axis is involved in ERK1/2 signaling and could mediate the migration and invasion of BC cells by regulating MMP13 activation. This study suggests that CCR4 might represent a promising prognostic biomarker and a potential therapeutic option for BC.     

Agonist-induced endocytosis of CC chemokine receptor 5 is clathrin dependent

The signaling activity of several chemokine receptors, including CC chemokine receptor 5 (CCR5), is in part controlled by their internalization, recycling, and/or degradation. For CCR5, agonists such as the chemokine CCL5 induce internalization into early endosomes containing the transferrin receptor, a marker for clathrin-dependent endocytosis, but it has been suggested that CCR5 may also follow clathrin-independent routes of internalization. Here, we present a detailed analysis of the role of clathrin in chemokine-induced CCR5 internalization. Using CCR5-transfected cell lines, immunofluorescence, and electron microscopy, we demonstrate that CCL5 causes the rapid redistribution of scattered cell surface CCR5 into large clusters that are associated with flat clathrin lattices. Invaginated clathrin-coated pits could be seen at the edge of these lattices and, in CCL5-treated cells, these pits contain CCR5. Receptors internalized via clathrin-coated vesicles follow the clathrin-mediated endocytic pathway, and depletion of clathrin with small interfering RNAs inhibits CCL5-induced CCR5 internalization. We found no evidence for CCR5 association with caveolae during agonist-induced internalization. However, sequestration of cholesterol with filipin interferes with agonist binding to CCR5, suggesting that cholesterol and/or lipid raft domains play some role in the events required for CCR5 activation before internalization.     

Chemokine Receptor CCR8 Is Required for Lipopolysaccharide-Triggered Cytokine Production in Mouse Peritoneal Macrophages

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8^{- /-}) and wild-type (WT) mice. We found that CCR8^-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca²⁺ flux and CCL1-driven PMφ aggregation. Similar to CCR8^-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8^-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8^-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8^-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.     

CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1-infected individuals

The basis for the extensive variability seen in the reconstitution of CD4(+) T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4(+) T cells/mm(3). The CCL3L1-CCR5 genotypes favoring CD4(+) T cell recovery are similar to those that blunted CD4(+) T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4(+) cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution.     

CCL5-CCR5-mediated apoptosis in T cells: Requirement for glycosaminoglycan binding and CCL5 aggregation

CCL5 (RANTES (regulated on activation normal T cell expressed and secreted)) and its cognate receptor, CCR5, have been implicated in T cell activation. CCL5 binding to glycosaminoglycans (GAGs) on the cell surface or in extracellular matrix sequesters CCL5, thereby immobilizing CCL5 to provide the directional signal. In two CCR5-expressing human T cell lines, PM1.CCR5 and MOLT4.CCR5, and in human peripheral blood-derived T cells, micromolar concentrations of CCL5 induce apoptosis. CCL5-induced cell death involves the cytosolic release of cytochrome c, the activation of caspase-9 and caspase-3, and poly(ADP-ribose) polymerase cleavage. CCL5-induced apoptosis is CCR5-dependent, since native PM1 and MOLT4 cells lacking CCR5 expression are resistant to CCL5-induced cell death. Furthermore, we implicate tyrosine 339 as a critical residue involved in CCL5-induced apoptosis, since PM1 cells expressing a tyrosine mutant receptor, CCR5Y339F, do not undergo apoptosis. We show that CCL5-CCR5-mediated apoptosis is dependent on cell surface GAG binding. The addition of exogenous heparin and chondroitin sulfate and GAG digestion from the cell surface protect cells from apoptosis. Moreover, the non-GAG binding variant, (44AANA47)-CCL5, fails to induce apoptosis. To address the role of aggregation in CCL5-mediated apoptosis, nonaggregating CCL5 mutant E66S, which forms dimers, and E26A, which form tetramers at micromolar concentrations, were utilized. Unlike native CCL5, the E66S mutant fails to induce apoptosis, suggesting that tetramers are the minimal higher ordered CCL5 aggregates required for CCL5-induced apoptosis. Viewed altogether, these data suggest that CCL5-GAG binding and CCL5 aggregation are important for CCL5 activity in T cells, specifically in the context of CCR5-mediated apoptosis.