No abstract

RANTES1 (regulated on activation, normal T cell expressed and secreted) and MIP (macrophage inflammatory protein)-1␣ are potent chemoattractant cytokines (chemokines) for T cells (1-3). Accumulating evidence suggests that these CC or ␤-chemokines function as regulators of inflammatory and immunoregulatory processes (4 -15). Chemokines mediate their shared and different biologic effects through common receptors. Eight receptor cDNAs specific for the CC chemokines have been cloned to date, whose nine gene products share a seven transmembrane domain architecture linked to G-protein complexes (16 -19). A number of studies have examined chemokine-induced signal transduction, yet defined signaling pathways have not been elaborated. Interleukin-8, the main chemotactic cytokine for neutrophils, will stimulate serine/threonine protein kinases (20). Monocyte chemotactic protein (MCP)-1 will activate p42/44 mitogen-activated protein kinases (21). MCP-1-, MCP-2-, and MCP-3-induced monocyte chemotaxis can be blocked by both serine/threonine and tyrosine kinase inhibitors (22). MCP-1 and MCP-3 rapidly induce arachidonic acid in target monocytes (23). RANTES, MIP-1␣, and MCP-1, -2, and -3 have been shown to promote Ca 2ϩ mobilization in monocytes, eosinophils, basophils, and T cells (7, 21, 24 -27).Many cytokines and growth factors mediate their effects via activation of a common signal transduction pathway, the STAT pathway. Binding of the ligand to its specific transmembrane receptor results in receptor aggregation, which may involve single or multiple receptor chains. Receptor aggregation leads to the catalytic activation of receptor-associated cytoplasmic protein-tyrosine kinases, termed janus kinases (JAK) and phosphorylation-activation of latent monomeric signal transducers and activators of transcription, Stat proteins. Six Stat proteins have been identified to date. Receptor-associated phosphorylated Stats then dimerize via SH2-phosphotyrosyl interactions and translocate to the nucleus, where they bind to specific promoter sequences, thereby regulating gene expression (reviewed in Ref. 29). All Stat proteins, with the possible exception of Stat2, differentially bind to more than ten related DNA elements, which fit the consensus TTNNNNNAA (consensus STAT recognition element). Conserved structural motifs within the cytoplasmic domains among the cytokine receptors have been implicated as Jak and STAT recognition sites (30, 31). Although Jak-STAT signaling is likely a common feature of all cytokines, recent reports suggest that STAT activation is not necessarily exclusively mediated via Jak association with the cytoplasmic domains of receptors that constitute the cytokine receptor superfamily. Angiotensin II binding to its cognate seven transmembrane, G-protein-coupled receptor, activates Stat1, Stat2,.Apart from their potent chemotactic activities, there is accumulating evidence that CC chemokines are also capable of stimulating T cells in vitro (35)(36)(37). At M concentrations, RANTES-induced signaling in T cells is med...

The chemokine RANTES (regulated on activation normal T cell expressed and secreted) and its cognate receptor CC chemokine receptor 5 (CCR5) have been implicated in regulating immune cell function. Previously we reported that in T cells, RANTES activation of CCR5 results in Stat1 and Stat3 phosphorylation-activation, leading to Stat1:1 and Stat1:3 dimers that exhibit DNA binding activity and the transcriptional induction of a Stat-inducible gene, c-fos. Given that RANTES and CCR5 have been implicated in T cell activation, we have studied RANTES-induced signaling events in a CCR5-expressing T cell line, PM1. RANTES treatment of PM1 T cells results in the rapid phosphorylation-activation of CCR5, Jak2, and Jak3. RANTES-inducible Jak phosphorylation is insensitive to pertussis toxin inhibition, indicating that RANTES-CCR5-mediated tyrosine phosphorylation events are not coupled directly to Galpha(i) protein-mediated events. In addition to Jaks, several other proteins are rapidly phosphorylated on tyrosine residues in a RANTES-dependent manner, including the Src kinase p56(lck), which associates with Jak3. Additionally our data confirm that the amino-terminally modified RANTES proteins, aminooxypentane-RANTES and Met-RANTES, are agonists for CCR5 and induce early tyrosine phosphorylation events that are indistinguishable from those inducible by RANTES with similar kinetics. Our data also demonstrate that RANTES activates the p38 mitogen-activated protein (MAP) kinase pathway. This is evidenced by the rapid RANTES-dependent phosphorylation and activation of p38 MAP kinase as well as the activation of the downstream effector of p38, MAP kinase-activated protein (MAPKAP) kinase-2. Pharmacological inhibition of RANTES-dependent p38 MAP kinase activation blocks MAPKAP kinase-2 activity. Thus, activation of Jak kinases and p38 MAP kinase by RANTES regulates the engagement of multiple signaling pathways.

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 bloodderived 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, ( 44 AANA 47 )-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.