Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 1. Identifi cation of culture conditions that enhance long-term CLL cell survival. Freshly isolated mononuclear cells were purifi ed on Ficol before culturing in RPMI supplemented with 10% heat inactivated FCS at a cell concentration of 1 – 60 10 6 cells/mL. (A) A single patient sample was seeded at the indicated densities and cell survival followed till day 14 ( * p 0.0001). (B) PBMCs were cultured at 5 10 7 cells/mL for 4 weeks with 50% medium changes every 2 – 3 days, weekly or fortnightly. (C) CLL PBMCs were cultured in complete medium for 7 days before supernatants were incubated with the ChemiArray III membrane. Complete medium was used as a negative control to account for non-specifi c binding. Seven CLL patient-samples were tested with similar results. Supernatant was collected from CLL PBMC cultures at indicated time points and (D) CCL2 and (E) CXCL2 protein concentration determined using ELISA. Results are displayed as mean SD ( n 2).

Article Snippet: In vitro chemokine assays Th e eff ect of chemokines IL-6, IL-8, CXCL2 and CCL2 was examined by culturing CLL PBMCs or B cells isolated from healthy donors in the presence of recombinant human IL-6 (2.5 ng/mL), recombinant human IL-8 (2.5 ng/mL), recombinant human CXCL2 (3.0 ng/mL) or recombinant human CCL2 (2.5 ng/mL) (all from R&D Systems).

Techniques: Isolation, Concentration Assay, Cell Culture, Incubation, Membrane, Negative Control, Binding Assay, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 3. Eff ect of IL-6, IL-8, CXCL2 and CCL2 on CLL cell survival in vitro . (A) CLL PBMCs were cultured for 7 days either alone or in the presence of recombinant human IL-6 (2.5 ng/mL) or IL-8 (2.5 ng/mL). Data are displayed as mean SD ( n 5) and each experiment was performed in triplicate ( * p 0.05, * * p 0.01). (B) CLL PBMCs were cultured with the addition of recombinant human CXCL2 (3.0 ng/mL) or CCL2 (2.5 ng/mL). Data are displayed as mean SD ( n 5) and each experiment was performed in triplicate ( * p 0.05, * * p 0.01). (C) B cells isolated from healthy donors were incubated with CCL2 and CXCL2 as described above. (D) Measurement of cell viability by trypan blue exclusion following the addition of recombinant human IL-6, IL-8, CCL2 or CXCL2 in diff erent combinations. Data are represented as mean fold-change SD ( n 6, each performed in duplicate or triplicate; * p 0.05, * * p 0.01).

Article Snippet: In vitro chemokine assays Th e eff ect of chemokines IL-6, IL-8, CXCL2 and CCL2 was examined by culturing CLL PBMCs or B cells isolated from healthy donors in the presence of recombinant human IL-6 (2.5 ng/mL), recombinant human IL-8 (2.5 ng/mL), recombinant human CXCL2 (3.0 ng/mL) or recombinant human CCL2 (2.5 ng/mL) (all from R&D Systems).

Techniques: In Vitro, Cell Culture, Recombinant, Isolation, Incubation

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 4. Mechanism of chemokine eff ect in CLL in vitro culture. (A) CLL PBMCs were cultured in the presence of CCL2 (2.5 ng/mL) or CXCL2 (3 ng/mL), with or without the addition of anti-human CCL2 antibody, anti-human CXCL1/2/3/GRO pan-specifi c antibody, and/or an isoptype control antibody, all at a concentration of 1 μ g/mL. Data are displayed as mean SD and are representative of one patient, performed in triplicate. (B) Cell viability was examined after 14 days following weekly addition of CXCL2 and/or CCL2 or isotype control antibody (all at 1 μ g/mL). Data are displayed as mean SD ( n 5, each performed in duplicate or triplicate; * p 0.05). CLL PBMCs or CD19 selected CLL cells were examined for (C) CCL2 and (D) CXCL2 protein expression after 1 day and 1 week using ELISA. Data are displayed as mean SD ( n 2, each in triplicate; * p 0.05, * * p 0.01, * * * p 0.001).

Article Snippet: In vitro chemokine assays Th e eff ect of chemokines IL-6, IL-8, CXCL2 and CCL2 was examined by culturing CLL PBMCs or B cells isolated from healthy donors in the presence of recombinant human IL-6 (2.5 ng/mL), recombinant human IL-8 (2.5 ng/mL), recombinant human CXCL2 (3.0 ng/mL) or recombinant human CCL2 (2.5 ng/mL) (all from R&D Systems).

Techniques: In Vitro, Cell Culture, Control, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 5. Confocal microscopy detects CCL2 and CXCL2 in cultured CLL PBMCs. Macrophage lineage cells and T cells were examined in 14-day-old CLL PBMC cultures for the production of CCL2 and CXCL2 by immunofl uorescence staining of CD68 and CD3 (magnifi cation 63). CD68 positive macrophage cells were examined for (A) CXCL2 and (B) CCL2, respectively, and CD3 positive T cells were examined for (C) CXCL2 and (D) CCL2, respectively. Cells staining positive for CD68 and CD3 appear red, cells staining positive for CCL2 and CXCL2 appear green, and nuclear staining appears blue (DAPI).

Article Snippet: In vitro chemokine assays Th e eff ect of chemokines IL-6, IL-8, CXCL2 and CCL2 was examined by culturing CLL PBMCs or B cells isolated from healthy donors in the presence of recombinant human IL-6 (2.5 ng/mL), recombinant human IL-8 (2.5 ng/mL), recombinant human CXCL2 (3.0 ng/mL) or recombinant human CCL2 (2.5 ng/mL) (all from R&D Systems).

Techniques: Confocal Microscopy, Cell Culture, Staining

Journal: Leukemia & lymphoma

Article Title: CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cells in vitro.

doi: 10.3109/10428194.2012.672735

Figure Lengend Snippet: Figure 6. Accessory cells protect CLL cells from apoptosis in vitro . (A) CLL PBMCs or CD19 purifi ed CLL cells were cultured as described previously and cell viability determined after 14 days. Data are displayed as mean SD ( n 5, each performed in duplicate or triplicate; * * * p 0.001). (B) Cell viability was examined after 14 days on cultured CLL PBMCs or indicated cell subsets. Data shown are mean SD ( n 5, each performed in duplicate or triplicate; * p 0.05, * * p 0.01, * * * p 0.001). (C) CLL PBMCs or CD19 selected CLL cells were cultured in the presence of recombinant human CXCL2 (2.5 ng/mL) or CCL2 (2.5 ng/mL) and cell viability determined after 14 days. Data shown are mean SD ( n 4; * p 0.05). (D) CD19 selected cells were cultured either alone or in the presence of accessory cells (either co-cultured or separated by a transwell) and cell viability determined after 14 days using trypan blue exclusion. Data are displayed as mean SD ( n 3) and each experiment was performed in triplicate ( * * p 0.01, * * * p 0.001).

Article Snippet: In vitro chemokine assays Th e eff ect of chemokines IL-6, IL-8, CXCL2 and CCL2 was examined by culturing CLL PBMCs or B cells isolated from healthy donors in the presence of recombinant human IL-6 (2.5 ng/mL), recombinant human IL-8 (2.5 ng/mL), recombinant human CXCL2 (3.0 ng/mL) or recombinant human CCL2 (2.5 ng/mL) (all from R&D Systems).

Techniques: In Vitro, Cell Culture, Recombinant

Journal: Oncogenesis

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

doi: 10.1038/oncsis.2012.25

Figure Lengend Snippet: Characterization of CCL2 -expressing clones. CCL2 ELISA results for a positive control (RH-6), a negative control (OV-90), stable EV clones: OV-90:EV 2 , OV-90:EV 9 , and stable CCL2 -expressing clones: OV-90: CCL2 23 and OV-90: CCL2 49 ( a ). Semi-quantitative RT–PCR analysis of CCL2 performed on complementary (c) DNA samples prepared from the parental OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 and OV-90: CCL2 49 ( b ). The expression of 18S cDNA is shown for RNA quality. Light microscope photographs of the cell morphology of OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 and OV-90: CCL2 49 , arrows indicate representative cell morphology ( c ). Light microscope photographs are × 100 magnification. Cell viability by XTT assay of OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 and OV-90: CCL2 49 , representative results from one independent experiment that was performed in triplicate ( d ). Cell viability was measured over 8 days and cell culture media-only wells served as controls (OSE and OSE-G418).

Article Snippet: Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona).

Techniques: Expressing, Clone Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Quantitative RT-PCR, Light Microscopy, XTT Assay, Cell Culture

Journal: Oncogenesis

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

doi: 10.1038/oncsis.2012.25

Figure Lengend Snippet: In vitro characterization of the CCL2 -expressing clones. Light microscope photographs of OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 and OV-90: CCL2 49 spheroids on day 8 of the assay, edges of the hanging droplets are shown ( a ). Colony forming ability of OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 and OV-90: CCL2 49 ( b ). Magnified ( × 4) colonies formed by OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 and OV-90: CCL2 49 ( c ). Average number of colonies formed in colony formation assay, representative results from one independent experiment that was performed in triplicate ( d ). Wound healing assay of OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 and OV-90: CCL2 49 over a period of 5 days ( e ). Light microscope photographs are × 100 magnification.

Article Snippet: Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona).

Techniques: In Vitro, Expressing, Clone Assay, Light Microscopy, Colony Assay, Wound Healing Assay

Journal: Oncogenesis

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

doi: 10.1038/oncsis.2012.25

Figure Lengend Snippet: S.c. injection site in vivo tumourigenicity assay. Mean survival, in days, of mice injected with OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 or OV-90: CCL2 49 at s.c. injection sites with six mice per group, and four phosphate-buffered saline (PBS)-injected mice as injection controls ( a ). Final mean tumour volume per group ( b ). Mean tumour volume per group over time ( c ).

Article Snippet: Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona).

Techniques: Injection, In Vivo, Saline

Journal: Oncogenesis

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

doi: 10.1038/oncsis.2012.25

Figure Lengend Snippet: I.p. injection site in vivo tumourigenicity assay. Mean survival, in days, of mice injected with OV-90, OV-90:EV 2 , OV-90:EV 9 , OV-90: CCL2 23 or OV-90: CCL2 49 at i.p. injection sites with six mice per group, and four PBS-injected mice as injection controls ( a ). Kaplan–Meier survival curves comparing all groups ( b ).

Article Snippet: Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona).

Techniques: Injection, In Vivo

Journal: Oncogenesis

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

doi: 10.1038/oncsis.2012.25

Figure Lengend Snippet: CCL2 expression and hematoxylin and eosin staining of xenografts. Semi-quantitative RT–PCR analysis of endogenous CCL2 ( CCL2 ) and introduced, expression vector-derived, CCL2 ( Flag/CCL2 ) performed on cDNA samples prepared from a subset of xenografts obtained from both i.p. and s.c. injection sites ( a ). The expression of 18S cDNA is shown for RNA quality. Hematoxylin and eosin staining of OV-90, OV-90: CCL2 23 and OV-90: CCL2 49 xenografts obtained from both s.c. and i.p. injection sites at time of killing ( b ). Images were obtained from the OlyVIA image viewer (Olympus America Inc., Center Valley, PA, USA).

Article Snippet: Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona).

Techniques: Expressing, Staining, Quantitative RT-PCR, Plasmid Preparation, Derivative Assay, Injection

Journal: Oncogenesis

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

doi: 10.1038/oncsis.2012.25

Figure Lengend Snippet: Enriched biological term and biological function analysis of the 89 probe sets differentially expressed in the CCL2 -expressing clones. Significantly enriched GO biological process terms derived from the DAVID functional annotation classification tool, with P -values <0.05 classified as significant by a modified Fisher's exact test ( a ). Ingenuity pathway analysis of enriched biological functions ( b ).

Article Snippet: Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona).

Techniques: Expressing, Clone Assay, Derivative Assay, Functional Assay, Modification

Journal: Oncogenesis

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

doi: 10.1038/oncsis.2012.25

Figure Lengend Snippet: CCL2 expression in HGOSC. CCL2 expression in 11 NOSE samples, normal whole ovary and 79 HGOSC samples as determined by Ziplex Research System expression array (Axela Inc., Toronto, Ontario, Canada) ( a ). Examples of CCL2 expression in cores from four HGOSC samples (left panel) showing, negative, detectable but low, moderately detectable and clearly detectable CCL2 staining, and examples of CCL2 staining in cores from two normal fallopian tube tissue samples (right panel) ( b ). Percentages refer to the proportion of the 187 HGOSC samples showing negative, detectable but low, moderately detectable and clearly detectable staining patterns of protein expression . ‘E' indicates epithelial cells and ‘S' indicates stromal cells. Immunohistochemistry images were obtained from the OlyVIA image viewer (Olympus America Inc.). The Kaplan–Meier survival curve analysis of HGOSC cases for overall ( c ) and disease-free ( d ) survival (in months) of patients whose tumours showed negative or low CCL2 staining ( n =136) compared with patients whose tumours showed moderately or clearly detectable CCL2 staining ( n =51). Indicated P -values were derived from log-rank tests.

Article Snippet: Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona).

Techniques: Expressing, Staining, Immunohistochemistry, Derivative Assay

Journal: Oncogenesis

Article Title: Overexpressing the CCL2 chemokine in an epithelial ovarian cancer cell line results in latency of in vivo tumourigenicity

doi: 10.1038/oncsis.2012.25

Figure Lengend Snippet: Summary of immunohistochemistry analysis of CCL2 in HGOSC samples

Article Snippet: Staining was performed with an antibody against human CCL2 (1 : 100; R&D Systems, Burlington, Ontario, Canada), using the Ventana Benchmark XT system (Ventana Medical Systems, Inc., Tucson, Arizona).

Techniques: Immunohistochemistry, Staining

Journal: Oncotarget

Article Title: Host genotype and tumor phenotype of chemokine decoy receptors integrally affect breast cancer relapse.

doi: 10.18632/oncotarget.4470

Figure Lengend Snippet: Figure 2: Tumor expression of chemokine decoy receptors (CDR) and host genotype of CDR jointly affect breast cancer relapse. A. Effect of tumor phenotype of CDR on relapse-free survival (RFS). P for log rank = 7.5 × 10−6. The RFS curve was derived from the Kaplan-Meier estimate, and the survival differences between groups were compared by log-rank test. B. Effect of host genotype of CDR on RFS. P for log rank = 0.002 C. Joint effect of tumor phenotype and host genotype of CDR on RFS. P-values of the differences between high expression/minor genotype group and high expression/major genotype group, high expression/major genotype group and low expression/ minor genotype group, and low expression/minor genotype group and low expression/major genotype group are 0.007, 0.354, and 0.047, respectively. High expression indicates co-expression of DARC and D6, otherwise low expression. Minor genotype indicates patients with at- least-one protective minor allele, otherwise major genotype. D. Chemokine levels in the supernatant of cells detected by ELISA after 24-hour incubation. For transient transfection, 1 μg pDARC-42G or -42A, 1 μg pD6-373S or -373Y, or the combination of 1 μg variant-type DARC- 42A and 1 μg variant-type pD6-373Y were transfected. An empty expression vector was also used as a control. 72 hours after transfection, the levels of human CCL2 and CCL5 in cell supernatants were determined with a sandwich ELISA. Columns represent the mean of three independent experiments; bars, standard error; *, P < 0.05. E. ROC curves assessing the discriminatory performance of the CDR phenotype/ genotype model and the CDR phenotype model for the prediction of disease relapse. P = 0.02 for AUC comparison.

Article Snippet: After 72 hours of transfection, the levels of human CCL2 and CCL5 in cell supernatants were determined with a sandwich ELISA (R&D systems, USA).

Techniques: Expressing, Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation, Transfection, Variant Assay, Plasmid Preparation, Control, Sandwich ELISA, Comparison

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 1. Exposure to high altitude results in PH and increased secretion of inflammatory classical monocyte ligands from the lungs. (A) Schematic showing hypoxia exposure time course in wildtype mice. Duration of hypoxia exposure is directly proportional to (B) RVSP and RV hypertrophy as measured by Fulton Index (N=6-13/group). At 3 days of hypoxia, increased protein expression of classical monocyte ligands (C) CCL2 (N=6-11/group) and (D) CCL12 (N=6- 11/group), whereas significantly lower levels of nonclassical monocyte ligand (E) CX3CL1 (N=6/group) in the lungs. (F) Higher CCL2 gradient in lungs and in the (G) peripheral blood of wildtype mice following 3 days of hypoxia exposure (N=5/group). Data in all panels were obtained from female mice. Statistical analysis was conducted using ANOVA, followed by Tukey's post hoc test. *P<0.05, **P<0.01, ****P<0.0001. N=number of animals, mean±SD, CI=confidence interval.

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Expressing

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 4: Genetic and pharmacologic blockade of CCR2-CCL2 axis protects from hypoxic PH. (A) Schematic showing the BM reconstitution of Ccr2-/- and WT BM into lethally irradiated wildtype mice. Wildtype mice reconstituted with Ccr2-/- BM were protected from hypoxic PH by attenuated (B) RVSP (N=7-11/group) and (C) RV hypertrophy (N=7-11/group) as measured by Fulton Index, compared to wildtype mice that were reconstituted with wildtype BM. (D) Schematic showing pharmacological blockade of CCR2 ligands CCL2 or CCL7 using anti-CCL2 or anti-CCL7 neutralizing antibody treatment. Hypoxia exposed wildtype mice treated with CCL2 NAb but not CCL7 NAb showed lower (E) RVSP (N=6/group) and (F) RV hypertrophy (N=6/group). TSP-1 levels in (G) lungs (N=6/group) and (H) blood (N=6/group); and TGF-β1 levels in (I) lungs (N=6/group) and (J) blood (N=6/group) compared to wildtype mice treated with isotype control antibody. Data in all panels followed a normal distribution. ANOVA with the Tukey test was performed for multiple comparisons. Data were obtained from the female mice. mean ± SD

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Irradiation, Control

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 5: Resident IMs are a major source of CCL2 and recruited IMs are a major source of pathologic TSP-1 in hypoxic PH. (A) Flow cytometry analysis using Ccl2RFP-flox reporter mice showed a higher number of CCL2+ IMs (N=14/group; N=14/group, 9F and 5M in Nx; 8F and 6M in Hx), and (B) FOLR2+ IMs are a major source of CCL2 (N=14/group). (C) Hypoxia exposed wildtype mice following intracellular CCL2 staining by flow cytometry also showed a higher number of CCL2+ IMs (N=7/group, female mice). (D). IM subpopulation analysis using flow

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Flow Cytometry, Staining

Journal: Journal of Clinical Investigation

Article Title: Monocytes and interstitial macrophages contribute to hypoxic pulmonary hypertension

doi: 10.1172/jci176865

Figure Lengend Snippet: Figure 8: DEX prophylaxis blunts CCL2 production by resident IMs and blocks the recruitment of TSP-1 producing CCR2+ IMs in hypoxia. (A) DEX prophylactically-treated, hypoxia-exposed Ccl2RFP-flox reporter mice exhibited a significant reduction in CCL2+ IMs, particularly in (B) CCL2RFP+ resident IMs (N=7/group). Additionally, (C) intracellular CCL2 flow cytometry analysis in DEX prophylactically-treated hypoxia-exposed wildtype mice revealed a

Article Snippet: Neutralizing Antibody and Pharmacological Treatment: Neutralizing mouse antibodies against CCL2 (Clone: 2H5; Cat.# BE0185), CCL7 (R&D System; Cat.# AF-456-NA), and isotype control (Cat.# BE0091; BioXCell, West Lebanon, NH, USA) were reconstituted in phosphatebuffered saline (PBS).

Techniques: Flow Cytometry

Journal: Frontiers in immunology

Article Title: Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

doi: 10.3389/fimmu.2021.638676

Figure Lengend Snippet: FIGURE 1 | DcR3.Fc- or HBD.Fc-treated M-Mφ and GM-Mφ secreted low amount of interleukin-1β (IL-1β) under particle stimulation. Six-day-cultured M-Mφ (A,C) and GM-Mφ (B) were derived from wild-type (WT) B6 and cocultured with hIgG (3 µg/ml), DcR3.Fc (3 µg/ml), or HBD.Fc (3 µg/ml). Cells were primed with lipopolysaccharides (LPS; 100 ng/ml) for 4 h and treated with monosodium urate (MSU) crystal, alum, or silica (150 or 300 µg/ml) for 6 h or ATP (3 mM) for 1 h. The concentration of IL-1β was measured by ELISA. All data shown were mean ± SD from three independent experiments. The statistical significance was determined by one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 were obtained by comparing the DcR3.Fc- or HBD.Fc-treated group to the hIgG-group. “NS” means no statistical significance.

Article Snippet: MouseM-CSF, mouse GM-CSF, human caspase-1, andmouse IL1β, IL-6, CCL2, CXCL2, and CXCL1 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

doi: 10.3389/fimmu.2021.638676

Figure Lengend Snippet: FIGURE 2 | DcR3 suppressed MSU-induced caspase-1 activation but did not affect the expression of NLRP3 or pro-IL-1β in LPS-treated macrophages. M-Mφ (A,C) and GM-Mφ (B,C) were treated with hIgG, DcR3.Fc, or HBD.Fc (3 µg/ml for each) for 6 days during differentiation stage in M-Mφ and GM-Mφ. Cells were treated with LPS (100 ng/ml) for 4 h, and total RNA was extracted. The mRNA levels of pro-IL-1β and NLRP3 were measured by real-time PCR (A,B). In some experiments, after LPS priming, cells were treated with MSU (300 µg/ml) for 3 h. The supernatants were harvested for caspase 1 p10 ELISA analysis (C). Data indicated mean ± SD of three independent experiments. The statistical significance was determined by one-way ANOVA. *p < 0.05 and ***p < 0.001 were obtained by comparing DcR3.Fc- or HBD.Fc-treated group to hIgG-group.

Article Snippet: MouseM-CSF, mouse GM-CSF, human caspase-1, andmouse IL1β, IL-6, CCL2, CXCL2, and CXCL1 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: Shared pathways significantly upregulated in endothelial cells treated with EVs.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Protein-Protein interactions, Migration, Chemotaxis Assay, Variant Assay, Activity Assay, Activation Assay

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: Endothelial cells were left untreated or treated for 16 hrs with LPS (1μg/mL) or EVs derived from non-infected or Mtb -infected macrophages. (A) Cells were stained with FITC conjugated anti-mouse TLR2 antibody or FITC conjugated anti-mouse IgG1 antibody as an isotype control. (B) Cells were surface-stained with FITC-labeled rat anti-mouse VCAM1 or FITC labeled anti-rat IgG2a antibody as an isotype control. (C) Cells were permeabilized and stained for intracellular CCL2 using PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gates were set to approximately 1% for isotype control and were maintained for all subsequent analysis. RC: untreated cells, RvEV: Treatment with EVs from H37Rv-infected macrophages, UnEV: Treated with EVs from non-infected macrophages. Data is representative of the protein expression from three independent experiments.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Derivative Assay, Infection, Staining, Control, Labeling, Expressing

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: (A) SVEC4-10 cell monolayers were left untreated or stimulated for 3 hrs with EVs derived from non-infected or Mtb -infected mice. CFSE-labeled mouse BMMs were added to SVEC4-10 cells. The fluorescently-labeled macrophages which migrated through the SVEC4-10 cell monolayer into the bottom of the Transwell filter were imaged. The number of BMMs in seven randomly selected fields were counted and the total number of cells for each condition defined. The data is the average of three independent mouse Mtb infections +SD with (*) indicating a p value < 0.05 compared to RC. (B) Quantitative RT-PCR was performed on endothelial cells that were left untreated or treated for 4 hours with EVs derived from uninfected or Mtb -infected macrophages. Total RNA was extracted followed by cDNA synthesis. Fold change of gene expression was calculated by comparative Ct method. Data is from two independent mouse Mtb infections. (C) Scatter plots of flow cytometry analysis of CCL2 expression. Endothelial cells were left untreated or treated for 16 hours with EVs derived from non-infected or Mtb -infected macrophages. Permeabilized cells were stained with PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gate was set for isotype control and was maintained for all subsequent analysis. RC: untreated cells. Un-EV: serum-derived EVs from uninfected mice, D7-EV, D14-EV, D21-EV: serum derived EVs from mice infected for 7, 14 and 21 days respectively. Data is representative of the CCL2 expression from two independent experiments.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Derivative Assay, Infection, Labeling, Quantitative RT-PCR, cDNA Synthesis, Gene Expression, Flow Cytometry, Expressing, Staining, Control