CCL5 promotes proliferation of MCF-7 cells through mTOR-dependent mRNA translation

The proliferative capacity of cancer cells is regulated by factors intrinsic to cancer cells and by secreted factors in the microenvironment. Here, we investigated the proto-oncogenic potential of the chemokine receptor, CCR5, in MCF-7 breast cancer cell lines. At physiological levels, CCL5, a ligand for CCR5, enhanced MCF-7.CCR5 proliferation. Treatment with the mTOR inhibitor, rapamycin, inhibited this CCL5-inducible proliferation. Because mTOR directly modulates mRNA translation, we investigated whether CCL5 activation of CCR5 leads to increased translation. CCL5 induced the formation of the eIF4F translation initiation complex through an mTOR-dependent process. Indeed, CCL5 initiated mRNA translation, shown by an increase in high-molecular-weight polysomes. Specifically, we show that CCL5 mediated a rapid up-regulation of protein expression for cyclin D1, c-Myc and Dad-1, without affecting their mRNA levels. Taken together, we describe a mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs, thereby providing CCR5-positive breast cancer cells with a proliferative advantage.     

Small molecule antagonists of CCR8 inhibit eosinophil and T cell migration

In this study, we demonstrate that in addition to T lymphocytes, human naïve eosinophils and the differentiated eosinophil-like cell line, AML14.3D10 express CCR8 and respond to CCL1 through CCR8 engagement. The responsiveness of cells was dependent on maturation stage, since CCL1 induced pronounced chemotaxis only in differentiated CCR8 positive AML14.3D10 cells. Despite the low CCR8 surface expression, human naïve eosinophils respond with a chemotaxis to high concentration CCL1. We further describe that Th2 clones in a maturation dependent fashion produce autocrine CCL1, which renders them unresponsive to further stimulation. An innovative method to enrich primary CCR8 reactive T cells was developed which demonstrates that primary peripheral CCR8 expressing T cells respond significantly to CCL1.We have developed novel small molecule CCR8 antagonists that are effective in inhibiting calcium mobilization and chemotaxis in differentiated AML cells as well as in human primary CCR8 positive T cells. Importantly, we demonstrate that the compounds can be divided into two subgroups: (i) compounds that are functional agonists for calcium mobilization and chemotaxis (ii) compounds that are pure antagonists. We demonstrate that agonism of these compounds does not correlate with their antagonistic potency. Taken together, we have identified a novel set of CCR8 compounds with antagonistic properties that inhibit CCL1 driven chemotaxis in both CCR8 expressing eosinophils as well as primary human T cells.     

Secretion of MIP-1β and MIP-1α by CD8(+) T-lymphocytes correlates with HIV-1 inhibition independent of coreceptor usage

CD8⁺ T-lymphocytes can utilize noncytolytic mechanisms to suppress HIV-1 replication through the secretion of soluble factors. The secretion of MIP-1β, MIP-1α, IP-10, MIG, IL-1α, and interferon gamma correlated most strongly with soluble noncytolytic suppression (p < 0.0001). Since the noncytolytic response is impaired by histone hyperacetylation, we examined the ability of histone hyperacetylation to alter the expression of immune-related genes. MIP-1α and IP-10 were also among the genes that were down-regulated by histone hyperacetylation. We define a multifactorial cytokine profile of CD8⁺ T-lymphocytes capable of mediating noncytolytic suppression of CXCR4-tropic HIV-1 replication.     

Heterophilic chemokine receptor interactions in chemokine signaling and biology

It is generally accepted that G-protein coupled receptors (GPCR), like chemokine receptors, form dimers or higher order oligomers. Such homo- and heterophilic interactions have been identified not only among and between chemokine receptors of CC- or CXC-subfamilies, but also between chemokine receptors and other classes of GPCR, like the opioid receptors. Oligomerization affects different aspects of receptor physiology, like ligand affinity, signal transduction and the mode of internalization, in turn influencing physiologic processes such as cell activation and migration. As particular chemokine receptor pairs exert specific modulating effects on their individual functions, they might play particular roles in various disease types, such as cancer. Hence, chemokine receptor heteromers might represent attractive therapeutic targets. This review highlights the state-of-the-art knowledge on the technical and functional aspects of chemokine receptor multimerization in chemokine signaling and biology.     

Chemokine oligomerization and interactions with receptors and glycosaminoglycans: the role of structural dynamics in function

The first chemokine structure, that of IL-8/CXCL8, was determined in 1990. Since then, many chemokine structures have emerged. To the initial disappointment of structural biologists, the tertiary structures of these small proteins were found to be highly conserved. However, they have since proven to be much more interesting and diverse than originally expected. Somewhat like lego blocks, many chemokines oligomerize and there is significant diversity in their oligomeric forms and propensity to oligomerize. Chemokines not only interact with receptors where different oligomeric forms can induce different signaling responses, they also interact with glycosaminoglycans which can stabilize oligomers and other structures that would not otherwise form in solution. Although chemokine monomers and dimers yielded quickly to structure determination, structural information about larger chemokine oligomers, chemokines receptors, and complexes of chemokines with glycosaminoglycans and receptors has been more difficult to obtain, but recent breakthroughs suggest that this information will be forthcoming, especially with receptor structures. Equally important and challenging, will be efforts to correlate the structural information with function.     

Chemokines and tuberculosis

Mycobacterium tuberculosis is a respiratory pathogen responsible for tuberculosis. A primary pathologic feature of M. tuberculosis infection is the formation of a granuloma. Immune cells migrate to the lung and then through the lung to the site of infection to form a granuloma. This structure contains the infection, and is often maintained for a long period of time. The signals responsible for granuloma formation and maintenance are largely unknown. Since chemokines and chemokine receptors direct cells to specific sites within the tissues, it is plausible that these cells participate in granuloma formation. In this review, the current literature on chemokines and M. tuberculosis infection, as well as the specific role that tumor necrosis factor alpha (TNF-) plays in granuloma formation and chemokine expression are discussed.     

Design and synthesis of novel small molecule CCR2 antagonists: Evaluation of 4-aminopiperidine derivatives

A novel N-(2-oxo-2-(piperidin-4-ylamino)ethyl)-3-(trifluoromethyl)benzamide series of human CCR2 chemokine receptor antagonists was identified. With a pharmacophore model based on known CCR2 antagonists a new core scaffold was designed, analogues of it synthesized and structure–affinity relationship studies derived yielding a new high affinity CCR2 antagonist N-(2-((1-(4-(3-methoxyphenyl)cyclohexyl)piperidin-4-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide.     

Defining the anti-inflammatory activity of a potent myxomaviral chemokine modulating protein,M-T7, through site directed mutagenesis

Viral chemokine modulating proteins provide new and extensive sources for therapeutics. Purified M-T7, a poxvirus-derived secreted immunomodulatory protein, reduces mononuclear cell invasion and atheroma in rodent models of angioplasty injury as well as aortic and renal transplant, improving renal allograft survival. M-T7 is a rabbit species-specific interferon gamma receptor (IFNγR) homolog, but also inhibits chemokine/glycosaminoglycan (GAG) interactions for C, CC and CXC chemokines, with cross-species specific inhibitory activity. M-T7 anti-atheroma activity is blunted in GAG deficient mouse aortic transplants, but not in CC chemokine receptor deficient transplants, supporting M-T7 interference in chemokine/GAG interactions as the basis of the atheroma-inhibitory activity. We have assessed point mutants of M-T7 both in vivo in a mouse angioplasty model and in vitro in tissue culture and binding assays, in order to better define the primary mechanism of anti-atheroma activity. Of these M-T7 mutants, the R¹⁷¹E and E²⁰⁹I M-T7 mutants lost inhibitory activity for plaque growth in hyperlipidemic ApoE^−/− mice after angioplasty injury and R¹⁷¹E, moreover, greatly exacerbated plaque growth and inflammation. F¹³⁷D retained some inhibitory activity for plaque growth. In contrast, for cell migration assays, M-T7-His_6X, F¹³⁷D, R¹⁷¹E, and E²⁰⁹I all inhibited CC chemokine (RANTES) mediated cell migration. For the ligand binding assays, R¹⁷¹E and E²⁰⁹I had significantly reduced binding to RANTES and IFNγ, whereas F¹³⁷D retained wild-type binding activity. Heparin treatment further reduced RANTES binding of all three M-T7 mutants. In summary, point mutations of M-T7, R¹⁷¹E and E²⁰⁹I, exhibited reduced anti-inflammatory properties in vivo after mouse angioplasty with a loss of in vitro binding to RANTES and IFNγ, indicating these point mutations partially disrupt M-T7 ligand-binding activities. Unexpectedly, the M-T7 mutants all retained inhibitory activity for human monocyte THP-1 cell migration ex vivo, suggesting additional inhibitory properties against human monocyte THP-1 cells that are independent of chemokine inhibition.     

Wnt5a Promotes Inflammatory Responses via Nuclear Factor κB (NF-κB) and Mitogen-activated Protein Kinase (MAPK) Pathways in Human Dental Pulp Cells

Wnt5a has been found recently to be involved in inflammation regulation through a mechanism that remains unclear. Immunohistochemical staining of infected human dental pulp and tissue from experimental dental pulpitis in rats showed that Wnt5a levels were increased. In vitro, Wnt5a was increased 8-fold in human dental pulp cells (HDPCs) after TNF-α stimulation compared with control cells. We then investigated the role of Wnt5a in HDPCs. In the presence of TNF-α, Wnt5a further increased the production of cytokines/chemokines, whereas Wnt5a knockdown markedly reduced cytokine/chemokine production induced by TNF-α. In addition, in HDPCs, Wnt5a efficiently induced cytokine/chemokine expression and, in particular, expression of IL-8 (14.5-fold) and CCL2 (25.5-fold), as assessed by a Luminex assay. The cytokine subsets regulated by Wnt5a overlap partially with those induced by TNF-α. However, no TNF-α and IL-1β was detected after Wnt5a treatment. We then found that Wnt5a alone and the supernatants of Wnt5a-treated HDPCs significantly increased macrophage migration, which supports a role for Wnt5a in macrophage recruitment and as an inflammatory mediator in human dental pulp inflammation. Finally, Wnt5a participates in dental pulp inflammation in a MAPK-dependent (p38-, JNK-, and ERK-dependent) and NF-κB-dependent manner. Our data suggest that Wnt5a, as an inflammatory mediator that drives the integration of cytokines and chemokines, acts downstream of TNF-α.