A new lineage of effector/memory CD4+ T cells has been identified whose signature products are IL‐17 cytokines and whose differentiation requires the nuclear receptor, RORγt. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL‐17 production for human T cells. Activating cord blood (naïve) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9 and CXCR6. Despite these data, we found no strong correlation between the production of IL‐17 and expression of CCR9 or CXCR6. By contrast, virtually all IL‐17‐producing CD4+ T cells, either made in our in vitro or found in peripheral blood, expressed CCR6. Compared with CD4+CD45RO+CCR6− cells, CD4+CD45RO+CCR6+ cells contained at least 100‐fold more IL‐17A mRNA and secreted 100‐fold more IL‐17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORγt. CCR6 was likewise expressed on all IL‐17‐producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL‐17‐producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL‐17‐related immune responses and possible targets for preventing inflammatory injury. Research support: NIAID, NIH

In this study, we examined the effects the prostacyclin receptor (IP) agonist cicaprost exhibited on U46619-mediated thromboxane A 2 receptor (TP) signaling in platelets and compared it to that which occurs in human embryonic kidney (

It is well established that leukocyte chemotactic receptors, a subset of G protein-coupled receptors, undergo endocytosis after stimulation by ligand. However, the significance of this phenomenon to cell motility and other important leukocyte functions induced by chemoattractants has not been clearly defined. Here we show that in primary human neutrophils, the threshold levels of agonist required for endocytosis of the chemotactic receptors CXCR1 and CXCR2 were ϳ10-fold or higher than those needed for maximal chemotactic and calcium flux responses. Moreover, when stimulated by agonists at concentrations that are high enough for chemotaxis but too low for receptor endocytosis, neutrophil CXCR1 and CXCR2 could be reactivated in response to repeated application of the same agonist. Both receptors were excluded from Triton X-100-insoluble lipid rafts, and at high agonist concentrations were rapidly endocytosed by a clathrin/rab5/dynamin-dependent pathway. These data support the conclusion that neutrophil migration in response to CXCR1 or CXCR2 agonists is not dependent on endocytosis of CXCR1 or CXCR2. Rather than being integral to the process of cell migration, receptor endocytosis may be a terminal stop signal when cells reach the focus of inflammation where the chemoattractant concentrations are the highest.

Here we demonstrate that the rate of ligand-induced endocytosis of CCR5 in leukocytes and expression systems is significantly slower than that of CXCR4 and requires prolonged agonist treatment, suggesting that these two receptors use distinct mechanisms. We show that the C-terminal domain of CCR5 is the determinant of its slow endocytosis phenotype. When the C-tail of CXCR4 was exchanged for that of CCR5, the resulting CXCR4-CCR5 (X4-R5) chimera displayed a CCR5-like trafficking phenotype. We found that the palmitoylated cysteine residues in this domain anchor CCR5 to plasma membrane rafts. CXCR4 and a C-terminally truncated CCR5 mutant (CCR5-KRFX) lacking these cysteines are not raft associated and are endocytosed by a clathrin-dependent pathway. Genetic inhibition of clathrin-mediated endocytosis demonstrated that a significant fraction of ligand-occupied CCR5 trafficked by clathrin-independent routes into caveolin-containing vesicular structures. Thus, the palmitoylated C-tail of CCR5 is the major determinant of its raft association and endocytic itineraries, differentiating it from CXCR4 and other chemokine receptors. This novel feature of CCR5 may modulate its signaling potential and could explain its preferential use by HIV for person-to-person transmission of disease. INTRODUCTIONClathrin-mediated endocytosis has been the general paradigm and the most extensively studied mechanism of ligand-induced receptor internalization (Schmid, 1997). However, in recent years, alternative clathrin-independent mechanisms have been described for the uptake of viruses, toxins, and receptors that lack conventional endocytic signals ). Among the latter mechanisms, cholesterol-rich structures called caveolae (Anderson, 1998;Kurzchalia and Parton, 1999) and lipid rafts have been implicated in diverse membrane processes including the assembly of signaling receptor complexes (Simons and Toomre, 2000). Endocytosis of some receptors such as the ␣ subunit of the IL-2 receptor (Tac antigen) and MHC-I, which lack canonical endocytic signals, follow distinct itineraries regulated by the small GTP binding protein, ADP-ribosylation factor 6 (Arf6; Radhakrishna and Donaldson, 1997;Sugita et al., 1999). Clathrin-independent endocytic itineraries are slower than the clathrin-dependent pathway and are functionally relevant in that the long residence time of occupied receptors at the cell surface may enable coupling to multiple signaling pathways.Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-11-0714. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E02-11-0714. † Corresponding author. E-mail address: aradhana@helix.nih.gov or sv1s@nih.gov. § Present address: INRS-Institut Armand-Frappier, Université du Québec, 531 des Prairies, Laval (Québec), Canada H7V 1B7. Abbreviations used: AOP-RANTES, aminooxypentane-regulated on activation, normal T-cell expressed and secreted; APC, allophycocyanin; Arf6, ADP-ribosylation factor 6; CCV, clathrin-coated vesicles; CHO, Chinese hamster o...