Thrombospondin-1-N-Terminal Domain Induces a Phagocytic State and Thrombospondin-1-C-Terminal Domain Induces a Tolerizing Phenotype in Dendritic Cells
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{"title"=>"Thrombospondin-1-N-terminal domain induces a phagocytic state and thrombospondin-1-C-terminal domain induces a tolerizing phenotype in dendritic cells", "type"=>"journal", "authors"=>[{"first_name"=>"Adi", "last_name"=>"Tabib", "scopus_author_id"=>"34870789300"}, {"first_name"=>"Alon", "last_name"=>"Krispin", "scopus_author_id"=>"14521091800"}, {"first_name"=>"Uriel", "last_name"=>"Trahtemberg", "scopus_author_id"=>"22136905200"}, {"first_name"=>"Inna", "last_name"=>"Verbovetski", "scopus_author_id"=>"6506378878"}, {"first_name"=>"Mario", "last_name"=>"Lebendiker", "scopus_author_id"=>"6603627984"}, {"first_name"=>"Tsafi", "last_name"=>"Danieli", "scopus_author_id"=>"6602422455"}, {"first_name"=>"Dror", "last_name"=>"Mevorach", "scopus_author_id"=>"55403634000"}], "year"=>2009, "source"=>"PLoS ONE", "identifiers"=>{"pui"=>"355219353", "sgr"=>"69949143845", "scopus"=>"2-s2.0-69949143845", "issn"=>"19326203", "pmid"=>"19721725", "doi"=>"10.1371/journal.pone.0006840"}, "id"=>"6844c8bd-9ed5-378f-b31a-858021115b7e", "abstract"=>"In our previous study, we have found that thrombospondin-1 (TSP-1) is synthesized de novo upon monocyte and neutrophil apoptosis, leading to a phagocytic and tolerizing phenotype of dendritic cells (DC), even prior to DC-apoptotic cell interaction. Interestingly, we were able to show that heparin binding domain (HBD), the N-terminal portion of TSP-1, was cleaved and secreted simultaneously in a caspase- and serine protease- dependent manner. In the current study we were interested to examine the role of HBD in the clearance of apoptotic cells, and whether the phagocytic and tolerizing state of DCs is mediated by the HBD itself, or whether the entire TSP-1 is needed. Therefore, we have cloned the human HBD, and compared its interactions with DC to those with TSP-1. Here we show that rHBD by itself is not directly responsible for immune paralysis and tolerizing phenotype of DCs, at least in the monomeric form, but has a significant role in rendering DCs phagocytic. Binding of TSP-1-C-terminal domain on the other hand induces a tolerizing phenotype in dendritic cells.", "link"=>"http://www.mendeley.com/research/thrombospondin1nterminal-domain-induces-phagocytic-state-thrombospondin1cterminal-domain-induces-tol", "reader_count"=>7, "reader_count_by_academic_status"=>{"Researcher"=>3, "Student > Doctoral Student"=>1, "Other"=>1, "Professor"=>1, "Student > Ph. D. Student"=>1}, "reader_count_by_user_role"=>{"Researcher"=>3, "Student > Doctoral Student"=>1, "Other"=>1, "Professor"=>1, "Student > Ph. D. Student"=>1}, "reader_count_by_subject_area"=>{"Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>2, "Medicine and Dentistry"=>1, "Psychology"=>1, "Chemistry"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Chemistry"=>{"Chemistry"=>1}, "Psychology"=>{"Psychology"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>2}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}}, "group_count"=>0}

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  • {"files"=>["https://ndownloader.figshare.com/files/884980"], "description"=>"<p>A. rHBD binds immature dendritic cells. iDCs were washed twice with RPMI, and were either incubated for 15 min on ice with 10 µg/ml rHBD or left untreated. Cells were then rewashed and stained with anti-HBD antibody (N20, Santa Cruz Biotechnology, Lot. F0804), fluorescence-labeled secondary antibody, as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006840#s2\" target=\"_blank\">Materials and Methods</a>, or fluorescence-labeled isotype control (p<0.00034). B and C. rHBD enhances apoptotic monocyte engulfment by iDCs. Apoptotic monocytes were stained with 5 µg/ml DiI and offered to iDCs, at a 4∶1 ratio. A control group of iDCs were not exposed to apoptotic monocytes. Acquisition of apoptotic cell-derived DiI by the DCs was measured after 8 h of interaction. iDCs were separated from monocytes based on DC-SIGN-FITC staining. Results are representative of three experiments. B. Comparison between the engulfment effect of rHBD and TSP-1 on DiI-stained apoptotic monocytes. Dot plot of DC-SIGN positive cells (iDCs) and DiI-stained apoptotic monocytes. Mean fluorescence of DiI acquisition is indicated in the right upper quadrant. Pre-incubation of the iDCs with 0.4 µg/ml HBD significantly enhanced apoptotic monocyte acquisition by iDCs; median fluorescence was 298 compared to 133 for iDCs exposed to apoptotic monocytes without addition of rHBD. HBD, at 1.0 µg/ml, further enhanced iDC engulfment capacity with median fluorescence 410. The effect of TSP-1, 2.0 µg/ml, is indicated for comparison, with median fluorescence 537. Results are representative of three experiments. See also <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006840#pone-0006840-g002\" target=\"_blank\">Figure 2C</a>. C. Comparison between the effects of rHBD from different sources (rHBD1, black bars; rHBD2, grey bars) on iDC uptake of apoptotic monocytes. Y axis represents the percentage increase in rHBD-dependent uptake of apoptotic monocytes by iDCs as measured by DiI fluorescence, compared with baseline uptake when no rHBD is added. A dose-dependent augmentation of uptake is seen with the addition of rHBD from both sources. Results are mean±SE of three experiments. D. Human monocyte-derived dendritic cell engulfing an apoptotic monocyte. Wide field fluorescence microscopy image of a human monocyte-derived dendritic cell that has engulfed a DiI-stained apoptotic monocyte (red). DAPI (blue) was used to demonstrate the nuclei, with an overlay of the phase contrast profile. Note the difference between the lightly fluorescent dendritic cell nucleus and the condensed and highly fluorescent chromatin of the apoptotic monocyte nucleus. Original magnification, 1000×. E. Isolated HBD enhances latex bead engulfment by iDCs. Green fluorescent latex beads were offered to iDCs at a 16∶1 ratio, in the presence or absence of 0–1 µg/ml isolated HBD or 2 µg/ml TSP-1. Median fluorescence was enhanced from 55.67 (average of two measurements) with no added HBD, to 81.33 in the presence of 0.4 µg/ml HBD, and 90.14 in the presence of 1 µg/ml HBD, indicating augmentation in phagocytic capacity of 46.08% and 61.9% respectively, following HBD exposure (p<0.001). This is compared with 98.38% augmentation of phagocytosis in the presence of 2 µg/ml TSP-1. Data is representative of three experiments.</p>", "links"=>[], "tags"=>["heparin", "binding", "thrombospondin-1", "enhances", "specific-", "nonspecific", "engulfment"], "article_id"=>555427, "categories"=>["Immunology", "Molecular Biology"], "users"=>["Adi Tabib", "Alon Krispin", "Uriel Trahtemberg", "Inna Verbovetski", "Mario Lebendiker", "Tsafi Danieli", "Dror Mevorach"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006840.g002", "stats"=>{"downloads"=>2, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_isolated_heparin_binding_domain_of_thrombospondin_1_enhances_the_specific_and_nonspecific_engulfment_capacity_of_iDCs_/555427", "title"=>"The isolated heparin binding domain of thrombospondin-1 enhances the specific- and nonspecific engulfment capacity of iDCs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 04:45:31"}
  • {"files"=>["https://ndownloader.figshare.com/files/884857"], "description"=>"<p>A. Fast protein liquid chromatography (FPLC) of heparin-agarose purified rHBD. HBD constructs were transformed into competent E. coli, amplified, verified, and extracted for expression in expressing bacteria (Origami B), as described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006840#s2\" target=\"_blank\">Materials and Methods</a>. Bacteria were lysed, and the rHBD was separated from the supernatant using FPLC equipped with a heparin-agarose column. The rHBD location was approximated using the FPLC fraction flow curve, as shown (left). The black line represents protein content of fractions eluted from the column. The peak containing the rHBD is marked. The dark gray line probably represents DNA remnants. SDS-PAGE served for rHBD identification. Clear, growing bands, located between 20–30 kDa, represent eluted rHBD. MW = molecular weight. Light gray line represents gradient B (elution salt gradient). B. Gel filtration. In order to obtain highly purified rHBD, FPLC protein-containing fractions were combined and separated using a molecular weight exclusion column. Protein content was determined using fraction-curve and SDS-PAGE. Black line = rHBD elution curve; Dark and light gray lines represent nonprotein side products. SDS-PAGE indicates the presence of a substantial amount of protein, located between 20–30 kDa. Control = rHBD; verified by mass spectrometry</p>", "links"=>[], "tags"=>["purification", "heparin", "binding"], "article_id"=>555305, "categories"=>["Immunology", "Molecular Biology"], "users"=>["Adi Tabib", "Alon Krispin", "Uriel Trahtemberg", "Inna Verbovetski", "Mario Lebendiker", "Tsafi Danieli", "Dror Mevorach"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006840.g001", "stats"=>{"downloads"=>1, "page_views"=>19, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Cloning_expression_and_purification_of_the_heparin_binding_domain_HBD_of_thrombospondin_1_/555305", "title"=>"Cloning, expression and purification of the heparin binding domain (HBD) of thrombospondin-1.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 04:44:47"}
  • {"files"=>["https://ndownloader.figshare.com/files/885157"], "description"=>"<p>A. Pre-incubation with HBD does not inhibit the expression of maturation-related molecules DR (upper chart) and CD86 (lower chart) when iDCs are exposed to LPS, whereas pre-incubation with TSP-1 at equimolar concentration inhibits their expression. Data is representative of three experiments. B. Compared to treatment with 10 ng/ml LPS, pre-incubation with different concentrations of HBD (0.4 or 1.0 µg/ml) before exposure to LPS did not have significant influence on expression of either DR (upper chart) or CD86 (lower chart). TSP-1, on the other hand, resulted in a decreased expression of both molecules, similar to that of untreated iDCs. Results are expressed as a percent of the expression of each molecule at the 10 ng/ml LPS treatment, and represent mean±SE of three experiments. Statistically significant difference is marked (*).</p>", "links"=>[], "tags"=>["rhbd", "tsp-1", "lps-stimulated"], "article_id"=>555610, "categories"=>["Immunology", "Molecular Biology"], "users"=>["Adi Tabib", "Alon Krispin", "Uriel Trahtemberg", "Inna Verbovetski", "Mario Lebendiker", "Tsafi Danieli", "Dror Mevorach"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006840.g004", "stats"=>{"downloads"=>1, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_rHBD_and_TSP_1_on_LPS_stimulated_iDCs_/555610", "title"=>"Effect of rHBD and TSP-1 on LPS-stimulated iDCs.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 04:46:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/885075"], "description"=>"<p>Immature DCs were pre-incubated with antibodies against CD29 or CD91, and were then offered apoptotic monocytes in the presence of 0.4 µg/ml recombinant HBD. HBD was also added to dendritic cells in the presence of an antibody raised against the HBD (N20). Striking (≥90%) inhibition of HBD-dependent apoptotic monocyte uptake is seen upon blocking HBD binding to either CD29 or CD91, compared with almost complete blocking of the HBD effect with the addition of N20. The effect of each blocking antibody on rHBD-dependent engulfment is measured relative to the difference in engulfment between additions of isotype control with 2 µg/ml HBD to that without HBD. Data is a mean±SE of three experiments.</p>", "links"=>[], "tags"=>["apoptotic", "monocyte", "engulfment", "mediated"], "article_id"=>555529, "categories"=>["Immunology", "Molecular Biology"], "users"=>["Adi Tabib", "Alon Krispin", "Uriel Trahtemberg", "Inna Verbovetski", "Mario Lebendiker", "Tsafi Danieli", "Dror Mevorach"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0006840.g003", "stats"=>{"downloads"=>2, "page_views"=>10, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_HBD_dependent_apoptotic_monocyte_engulfment_may_be_mediated_through_different_receptors_/555529", "title"=>"HBD-dependent apoptotic monocyte engulfment may be mediated through different receptors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 04:46:05"}

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