Anja Mueller

Antitumor Effects of the Mouse Chemokine 6Ckine/SLC Through Angiostatic and Immunological Mechanisms

Mouse 6Ckine/SLC (secondary lymphoid tissue chemokine) is a chemotactic factor for dendritic cells, T cells, and NK cells in vitro. In addition, mouse 6Ckine/SLC interacts with the chemokine receptor CXCR3, as do several chemokines with antiangiogenic properties. These dual properties of mouse 6Ckine/SLC were tested for the induction of an antitumor response by transducing the C26 colon carcinoma tumor cell line with a cDNA encoding mouse 6Ckine/SLC. The C26-6CK-transduced cells showed reduced tumorigenicity in immunocompetent or in nude mice. Part of this effect was likely due to angiostatic mechanisms as shown by immunohistochemistry and Matrigel assay. C26-6CK tumors were also heavily infiltrated with leukocytes, including granulocytes, dendritic cells, and CD8+ T cells. In vivo, anti-CD8 treatment increased the tumorigenicity of the C26-6CK tumor cells, and tumor-infiltrating CD8+ T cells had the phenotype of memory effector cells, suggesting the induction of cytotoxic tumor-specific T lymphocytes. On the other hand, anti-asialo-GM1 depletion also increased the tumorigenicity of C26-6CK cells, supporting the participation of NK cells. Finally, tumor-infiltrating dendritic cells had the phenotype and functional features of immature dendritic cells. Overall, these results suggest that mouse 6Ckine/SLC has strong antitumor effects by inducing both angiostatic, CD8+ T cell-mediated, and possibly NK-mediated tumor resistance mechanisms.

The chemokine receptor, CCR5

The chemokine receptor, CCR5, is a G protein coupled receptor responsible for some of the effects of the chemokines CCL3, CCL4 and CCL5. It is also one of the co-receptors for the entry of human immunodeficiency virus-1 (HIV-1) into cells. Regulation of CCR5 number on cells is, therefore, important for determining the infection rate by HIV-1.

The chemokine receptor CXCR3 is degraded following internalization and is replenished at the cell surface by de novo synthesis of receptor.

The chemokine receptor CXCR3 is expressed on the surface of both resting and activated T lymphocytes. We describe in this study the endocytosis of CXCR3 using T lymphocytes and CXCR3 transfectants. Chemokine-induced CXCR3 down-regulation occurred in a rapid, dose-dependent manner, with CXCL11 the most potent and efficacious ligand. Endocytosis was mediated in part by arrestins, but appeared to occur independently of clathrin and caveolae. In contrast to other chemokine receptors, which are largely recycled to the cell surface within an hour, cell surface replenishment of CXCR3 occurred over several hours and was dependent upon mRNA transcription, de novo protein synthesis, and transport through the endoplasmic reticulum and Golgi. Confocal microscopy and Western blotting confirmed the fate of endocytosed CXCR3 to be degradation, mediated in part by lysosomes and proteosomes. Site-directed mutagenesis of the CXCR3 C terminus revealed that internalization and degradation were independent of phosphorylation, ubiquitination, or a conserved LL motif. CXCR3 was found to be efficiently internalized in the absence of ligand, a process involving a YXXL motif at the extreme of the C terminus. Although freshly isolated T lymphocytes expressed moderate cell surface levels of CXCR3, they were only responsive to CXCL11 with CXCL9 and CXCL10 only having significant activity on activated T lymphocytes. Thus, the activities of CXCR3 are tightly controlled following mRNA translation. Because CXCR3+ cells are themselves a source of IFN-γ, which potently induces the expression of CXCR3 ligands, such tight regulation of CXCR3 may serve as a control to avoid the unnecessary amplification of activated T lymphocyte recruitment.

The CXC Chemokine MIG/CXCL9 Is Important in Innate Immunity against Streptococcus pyogenes.

Pharyngitis caused by Streptococcus pyogenes is one of the most common bacterial infections in humans and is also a starting point for invasive S. pyogenes infection. Here, we describe that tonsil fluid from patients with streptococcal pharyngitis contains high amounts of the interferon (IFN)-dependent CXC chemokine known as monokine induced by IFN- gamma (MIG)/CXCL9. Also in vitro, inflamed pharyngeal epithelium produced large amounts of MIG/CXCL9 in the presence of bacteria. The CXC chemokines MIG/CXCL9, IFN-inducible protein-10/CXCL10, and IFN-inducible T cell alpha -chemoattractant/CXCL11 all showed antibacterial activity against S. pyogenes, and inhibition of MIG/CXCL9 expression reduced the antibacterial activity at the surface of inflamed pharyngeal cells. S. pyogenes of the clinically important M1 serotype secrets the protein streptococcal inhibitor of complement (SIC), which inhibited the antibacterial activity of the chemokines. As exemplified by S. pyogenes pharyngitis, the data identify a novel innate defense mechanism against invasive bacteria on epithelial surfaces.

CXCL4-induced migration of activated T lymphocytes is mediated by the chemokine receptor CXCR3

The chemokine CXCL4/platelet factor‐4 is released by activated platelets in micromolar concentrations and is a chemoattractant for leukocytes via an unidentified receptor. Recently, a variant of the human chemokine receptor CXCR3 (CXCR3‐B) was described, which transduced apoptotic but not chemotactic signals in microvascular endothelial cells following exposure to high concentrations of CXCL4. Here, we show that CXCL4 can induce intracellular calcium release and the migration of activated human T lymphocytes. CXCL4‐induced chemotaxis of T lymphocytes was inhibited by a CXCR3 antagonist and pretreatment of cells with pertussis toxin (PTX), suggestive of CXCR3‐mediated G‐protein signaling via Gαi‐sensitive subunits. Specific binding by T lymphocytes of the CXCR3 ligand CXCL10 was not effectively competed by CXCL4, suggesting that the two are allotopic ligands. We subsequently used expression systems to dissect the potential roles of each CXCR3 isoform in mediating CXCL4 function. Transient expression of the CXCR3‐A and CXCR3‐B isoforms in the murine pre‐B cell L1.2 produced cells that migrated in response to CXCL4 in a manner sensitive to PTX and a CXCR3 antagonist. Binding of radiolabeled CXCL4 to L1.2 CXCR3 transfectants was of low affinity and appeared to be mediated chiefly by glycosaminoglycans (GAGs), as no specific CXCL4 binding was observed in GAG‐deficient 745‐Chinese hamster ovary cells stably expressing CXCR3. We suggest that following platelet activation, the CXCR3/CXCL4 axis may play a role in T lymphocyte recruitment and the subsequent amplification of inflammation observed in diseases such as atherosclerosis. In such a setting, antagonism of the CXCR3/CXCL4 axis may represent a useful, therapeutic intervention.

Cellular Uptake of a Fluorescent Calix[4]arene Derivative

A rapid synthetic route to a nontoxic fluorescently labeled water-soluble calixarene has been developed. Investigation of the cellular uptake of the labeled calixarene, via confocal microscopy, through coincubation with uptake inhibitors demonstrates that uptake is not through the common clathrin coated pits or caveolae (lipid raft) endocytic pathways and that the calixarene derivative localizes within the cytoplasm and does not enter the nucleus. The study demonstrates the power of fluorescent labeling for investigation of interactions between calixarenes and biological systems and the potential for calixarene based intracellular imaging agents.

Np9 Protein of Human Endogenous Retrovirus K Interacts with Ligand of Numb Protein X

ABSTRACT We have recently identified Np9 as a novel nuclear protein produced by the human endogenous retrovirus K and were able to document the exclusive presence of np9 transcript in tumors and transformed cells. With the aim of studying whether Np9 has a role in tumorigenesis, a systematic search for interacting proteins was performed. Here, we identify the RING-type E3 ubiquitin ligase LNX (ligand of Numb protein X) as an Np9-interacting partner. We furthermore show that the interaction involves N- and C-terminal domains of both proteins and can affect the subcellular localization of LNX. LNX has been reported to target the cell fate determinant and Notch antagonist Numb for proteasome-dependent degradation, thereby causing an increase in transactivational activity of Notch. We document that LNX-interacting Np9, like Numb, is unstable and degraded via the proteasome pathway and that ectopic Numb can stabilize recombinant Np9. Combined, these findings point to the possibility that Np9 affects tumorigenesis through the LNX/Numb/Notch pathway.

Pharmacological characterization of the chemokine receptor, CCR5

We investigated the effects of a number of naturally occurring chemokines (MIP‐1α, MIP‐1β, RANTES, MCP‐2, MCP‐3, MCP‐4) on different processes linked to the chemokine receptor CCR5 in recombinant CHO cells expressing the receptor at different levels. Internalization of CCR5 following chemokine treatment was studied and MIP‐1α, MIP‐1β and RANTES (50 nM) were able to induce internalization (∼50%) of the receptor. Internalization due to MCP‐2, MCP‐3 and MCP‐4 was less (∼20%). Phosphorylation of CCR5 following chemokine treatment was studied and MIP‐1α, MIP‐1β and RANTES (50 nM) were able to induce phosphorylation of CCR5 whereas the other chemokines did not induce CCR5 phosphorylation. MIP‐1α, MIP‐1β, RANTES and MCP‐2 were able to stimulate [35S]‐GTPγS binding, an index of receptor/G protein activation, whereas MCP‐3 and MCP‐4 had no effect in this assay. MCP‐2 was a partial agonist (∼80%) compared to MIP‐1α, MIP‐1β and RANTES, which gave similar maximal stimulations in this assay. MIP‐1α, MIP‐1β, RANTES, MCP‐2 and MCP‐4 were able to stimulate increases in intracellular calcium ions via activation of CCR5 whereas MCP‐3 was without effect. It is concluded that different chemokines interacting with CCR5 mediate different patterns of cellular responses.

CCL3, acting via the chemokine receptor CCR5, leads to independent activation of Janus kinase 2 (JAK2) and Gi proteins

The interaction of the chemokine receptor, CCR5, expressed in recombinant cells, with different G proteins was investigated and CCR5 was found to interact with Gi, Go and Gq species. Interaction with Gi leads to G protein activation, whereas Gq does not seem to be activated. Additionally, CCR5 activation also leads to phosphorylation of Janus kinase 2 (JAK2). Activation of JAK2 is independent of Gi or Gq activation. Gi protein activation was not prevented by inhibition of JAK, showing that heterotrimeric G protein activation and activation of the JAK/signal transducer and activator of transcription (STAT) pathway are independent of each other.

Synthesis of D-mannose capped silicon nanoparticles and their interactions with MCF-7 human breast cancerous cells.

Silicon nanoparticles (SiNPs) hold prominent interest in various aspects of biomedical applications. For this purpose, surface functionalization of the NPs is essential to stabilize them, target them to specific disease area, and allow them to selectively bind to the cells or the bio-molecules present on the surface of the cells. However, no such functionalization has been explored with Si nanoparticles. Carbohydrates play a critical role in cell recognition. Here, we report the first synthesis of silicon nanoparticles functionalized with carbohydrates. In this study, stable and brightly luminescent d-Mannose (Man) capped SiNPs have been synthesized from amine terminated SiNPs and d-mannopyranoside acid. The surface functionalization is confirmed by Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), and energy dispersive X-ray spectroscopy (EDX) studies. The mean diameter of the crystal core is 5.5 nm, as measured by transmission electron microscopy (TEM), while the hydrodynamic diameter obtained by dynamic light scattering (DLS) is 16 nm. The quantum yield (QY) of photoluminescence emission is found to be 11.5%, and the nanoparticles exhibit an exceptional stability over two weeks. The Man-capped SiNPs may prove to be valuable tools for further investigating glycobiological, biomedical, and material science fields. Experiments are carried out using Concanavalin A (ConA) as a target protein in order to prove the hypothesis. When Man functionalized SiNPs are treated with ConA, cross-linked aggregates are formed, as shown in TEM images as well as monitored by photoluminescence spectroscopy (PL). Man functionalized SiNPs can target cancerous cells. Visualization imaging of SiNPs in MCF-7 human breast cancer cells shows the fluorescence is distributed throughout the cytoplasm of these cells.