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First published online 17 October 2006
doi: 10.1242/jcs.03231

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HIV-1 Nef upregulates CCL2/MCP-1 expression in astrocytes in a myristoylation- and calmodulin-dependent manner

¹ Institute of Molecular Virology, GSF-National Research Center for Environment and Health, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany
² Institute of Virology, Technical University of Munich, Schneckenburgerstr. 8, 81675 Munich, Germany

View larger version (8K): [in a new window]   Fig. 1. Astrocytes expressing nef secrete a factor that is chemotactic for monocytes. A chemotaxis assay was performed using THP-1 cells. Cell culture supernatants from 16-hour incubations of astrocytic U251MG-parental (P) or stably transfected U251MG-NefBru (clone 2/8.2, 4/4.1 and 4/4.2) or U251MG-NefTH, or with the vector carrying the neomycin resistance gene (-pNeo) were placed in the lower part of the chemotaxis unit. Recombinant CCL2/MCP-1 (25 ng/ml) was used as a positive control stimulus. Monocytic THP-1 cells were added into the upper part and allowed to migrate for 2 hours into the lower part. Subsequently, cell numbers in the lower part were counted and chemotaxis indexes were calculated as described in the Material and Methods. Data represent mean ± s.e.m. from five independent experiments; *, P<0.05.

View larger version (54K): [in a new window]   Fig. 2. Nef modulates cytokine and growth factor production in astrocytes. U251MG-NefBru-4/4.2 and U251MG-parental cells were incubated in VLE-RPMI1640, 0.5% FCS for 16 hours. Supernatants were analyzed with RayBio® Human Cytokine antibody array V. (A) Scheme of the spotted primary antibodies. (B) Images after fluorescence scanning. (C) List of differentially regulated proteins. The ratios indicated are calculated by using the fluorescence intensities of the corresponding protein spots after background (Neg) correction and normalization of the intensities according to the mean intensities of the positive controls (Pos).

View larger version (22K): [in a new window]   Fig. 3. CCL2/MCP-1 expression is increased in nefBru-expressing U251MG cells. (A) Astrocytic U251MG-NefBru (clones 4/4.1 and 4/4.2), -NefTH, -pNeo and -parental cells were seeded at a density of 2x105 or 4x105 cells/ml, incubated for 8, 16, 24 or 36 hours, and supernatants were subsequently analyzed with CCL2/MCP-1 ELISA. Fold inductions were calculated relative to the CCL2/MCP-1 protein concentrations determined in the supernatants of U251MG-parental cells. Data represent mean ± s.e.m. from at least six independent experiments; *, P<0.05. (B) Kinetic analysis of CCL2/MCP-1 protein secretion. Cells were incubated for periods as indicated and CCL2/MCP-1 protein concentrations determined by ELISA in the cellular supernatants. One representative experiment is shown. Data represent mean ± s.e.m. of duplicates. (C) Cells were incubated for 4 hours in the presence of monensin and subsequently stained intracellularly with PE-labeled mAb to CCL2/MCP-1 (black outline) or PE-labeled isotype-matched control antibody (gray). The ratio of the mean fluorescence intensities from CCL2/MCP-1-stained to isotype-stained cells is indicated in the upper right corner of each plot. (D) Total RNA was isolated from astrocytic U251MG-NefBru (clones 2/8.2, 4/4.1 and 4/4.2), -NefTH and -pNeo cells, and analyzed by MultiprobeTM RNase protection assay using biotinylated hCK-5 as probe.

View larger version (10K): [in a new window]   Fig. 4. CCL2/MCP-1 is the exclusive factor in the culture supernatants of U251MG-NefBru cells capable of inducing chemotaxis of monocytic THP-1 cells. Cell culture supernatants from nefBru-expressing astrocytes (clones 4/4.1 or 4/4.2) or rCCL2/MCP-1 (25 ng/ml) were placed into the lower part of the chemotaxis unit and THP-1 cells in the upper part. At 45 minutes prior to the assay, supernatants and rCCL2/MCP-1-containing wells were incubated with anti-CCL2/MCP-1 antibodies or isotype-matched control antibodies as indicated. One representative experiment of four independent experiments is shown. Data represent mean ± s.e.m. of triplicates.

View larger version (19K): [in a new window]   Fig. 5. Supernatants of U251MG-NefBru cells do not induce chemotaxis of CCR2-deficient monocytic cells. (A) Immunocytochemistry analysis of monocytic THP-1 and U-937 cells using anti-CCR2 Ab. To exclude non-specific staining by the secondary antibody, anti-CCR2 Ab was substituted with distilled water (Control). Images were obtained using a microscope (Axiolab, Carl-Zeiss Jena) equipped with a x40 objective and a x10 ocular lenses. Original magnification, x100. (B) CCR2-specific RT-PCR with total RNA originating from THP-1 cells (T) and U-937 cells (U). GAPDH served as internal control. (C) Supernatants of U251MG-NefBru-4/4.2, -parental, -pNeo cells and rCXCL12/SDF-1 (25ng/ml) were placed into the lower parts of the chemotaxis unit, and U-937 cells in the upper parts. One representative experiment is shown. Data represent mean ± s.e.m. of triplicates.

View larger version (21K): [in a new window]   Fig. 6. Nef-induced CCL2/MCP-1 expression in astrocytes is dependent on calmodulin. U251MG-parental (P) and U251MG-NefBru cells (Nef) were treated with 50 µM W7 (+) or solvent (–) for 16 hours. Total RNA was isolated and analyzed with MultiprobeTM ribonuclease protection assay (A) and RT-PCR (B). The ratios of the CCL2/MCP-1 to GAPDH intensities were determined and the values obtained from the solvent-treated U251MG-Nef cells were set to 100%. One representative experiment of three independent experiments is shown. (C) Astrocytic U251MG-NefBru, -pNeo and -parental cells were incubated for 8 hours with W7 at concentrations indicated, and CCL2/MCP-1 protein in the supernatants was determined by ELISA. One representative experiment is shown. Data represent mean ± s.e.m. of duplicates.

View larger version (19K): [in a new window]   Fig. 7. Myristoylation-deficient NefBru does not increase CCL2/MCP-1 protein and mRNA production. U251MG-parental cells were transiently transfected with NefBru, NefBru-GG2AA or CAT. The cell culture supernatants were replaced with medium 8, 24 and 32 hours after transfection. (A) Alignment of the N-terminal amino acid sequence of NefBru and NefBru-GG2AA. (B). Simultaneous determination of Nef and GAPDH concentrations by western blotting. Cells were lysed after transfection as indicated. (C) CCL2/MCP-1 protein concentrations in the cell culture supernatants were determined using ELISA after transfection as indicated. Data represent mean ± s.e.m. of three separate transfection experiments. (D) Total RNA was isolated 48 hours after transfection and analyzed with RT-PCR. The ratios of the CCL2/MCP-1 to GAPDH intensities were determined and the values obtained from the NefBru-transfected cells were set to 100%.