Journal:

Article Title: Characterizing a soluble survival signal for activated lymphocytes from CD14 + cells

doi: 10.1046/j.1365-2567.2002.01463.x

Figure Lengend Snippet: Effects of CD14+ cell depletion on lymphocyte activation and proliferation. CD14+ peripheral blood mononuclear cells (PBMC) were isolated by Histopaque and depleted of CD14+ cells by Dynabeads (CD14− PBMC). CD14+ and CD14− PBMC were then stimulated with phytohaemagglutinin-p (PHA-p) (10 µg/ml) at a concentration of 1·0×106 cells/ml (a) CD14+ and CD14− PBMC were analysed by flow cytometry at 24, 48 and 72 hr for the expression of CD69 antigen. *P < 0·01: CD69/CD14− PBMC versus CD69/CD14+ PBMC at 24 and 72 hr. †P < 0·05: CD3/69/CD14− PBMC versus CD3/69/CD14+ PBMC at 72 hr. n = 10 (CD69); n = 5 (CD3/69); independent sample t-test. The differences of CD69 expression at 48 hr and the differences of CD3/69 expression at 24/48 hr were not significant (P > 0·05). Bars show the mean value±1 SE. (b) CD14+ and CD14− PBMC were analysed by flow cytometry for the expression of CD25, CD122 and CD132 antigens. *P < 0·01: CD14+ (24 hr) versus CD14−(24 hr); paired t-test, n = 8. CD25, CD122 and CD132 expression before and after CD14+ cell depletion at all the other time-points were not significant. Bars show the mean value±1 SE. (c) Supernatants from CD14+ and CD14− PBMC cultures were collected, aliquoted and stored at −80°. Enzyme-linked immunosorbent assay (ELISA) was used to test for interleukin-2 (IL-2) production. *P < 0·05; †P < 0·01: CD14+ versus CD14−; n = 8; paired sample t-test. Bars show the mean value±1 SE. (d) At 24, 48 and 72 hr, viable cells were counted as Trypan blue-excluded cells. Cell death was analysed by flow cytometry using annexin V-fluorescein isothiocyanate (FITC) and -propidium iodide (PI) staining, and by Trypan blue staining. The data represent the mean of three independent experiments. Hatched bars represent cell death rates and lines represent lymphocyte counts. CD14+/PI & Annexin and CD14−/PI & Annexin: cell death in CD14+ and CD14− PBMC stained with PI/Annexin and analysed by flow cytometry; CD14+/TB and CD14−/TB: cell death in CD14+ and CD14− PBMC stained with Trypan blue and analysed by light microscopy; CD14+/count and CD14−/count: viable lymphocyte count stained with viable excluding dye (Trypan blue) and analysed by light microscopy.

Article Snippet: IL-1β, TNF-α and IL-6 depletion assays IL-1β (catalogue no. DLB50; R & D Systems, Minneapolis, MN), TNF-α (catalogue no. DTA50; R & D Systems) and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (catalogue no. D6050; R & D Systems) were used.

Techniques: Activation Assay, Isolation, Concentration Assay, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Light Microscopy

Journal:

Article Title: Characterizing a soluble survival signal for activated lymphocytes from CD14 + cells

doi: 10.1046/j.1365-2567.2002.01463.x

Figure Lengend Snippet: CD14 cocktails protect activated lymphocytes from undergoing activation-induced cell death (AICD). Dynabeads were mixed with CD14+ peripheral blood mononuclear cells (PBMC) at a ratio of 50 µl/107 cells and incubated at 4° for 1 hr with gentle rotation. After 1 hr, free Dynabeads and Dynabeads-bound cells were collected using a magnetic particle concentrator (Dynal, MPC). The Dynabeads and Dynabeads-bound cells were resuspended in serum-free medium. (a) CD14 cocktails were collected at 18 hr after incubation of Dynabeads-bound cells (CD14+ cells) with Dynabeads. The interleukin (IL)-1β, tumour necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), IL-6, IL-2, IL-12 and IL-15 enzyme-linked immunosorbent assay (ELISA) kits were used to test the cytokine contents. Bars show the mean value±1 SE. (b) CD14+ PBMC, CD14− PBMC and CD14− PBMC with addition of different doses of CD14 cocktails were cultured at 37° in 5% CO2. At 0, 24 and 48 hr time-points, cells were analysed by flow cytometry and the apoptotic rates were quantified as propidium iodide (PI)-positive cells. The data represents the mean of three independent experiments. Bars show the mean value±1 SE. (c) CD14+ PBMC, CD14− PBMC and CD14− PBMC with addition of 20% CD14 cocktails were cultured at 37° in 5% CO2. At 24, 48 and 72 hr time-points, apoptosis was analysed as described in (b). *CD14+ versus CD14−: P < 0·001 at all time-points; ‡CD14− versus CD14−/CD14CK: P < 0·001 at all time-points; AICD rates between CD14+ and CD14−/CD14CK were not significant at any time-point. n = 12, analysis of variance (anova). Bars show the mean value ± 1 SE. (d) CD14+ and CD14− PBMC were stimulated with phytohaemagglutinin-p (PHA-p) at a concentration of 1×106 cells/ml. CD14 cocktails (20%, vol/vol) were added to some of the CD14− PBMC at time 0. At 24, 48 and 72 hr time-points, cell numbers were counted in each well. Viable cells were counted using Trypan blue staining. *CD14+ versus CD14−: P < 0·05 at 24 hr, P < 0·01 at 48 hr, P < 0·0001 at 72 hr; †CD14+ versus CD14−/CD14CK: P < 0·01 at 72 hr, not significant at 24 and 48 hr; ‡CD14− versus CD14−/CD14CK: P < 0·05 at 48 hr, P < 0·01 at 72 hr, not significant at 24 hr. n = 7, analysis of variance (anova). Bars show the mean value±1 SE. (e) Culture supernatants from CD14+ PBMC, CD14− PBMC and CD14− PBMC with addition of 20% CD14 cocktails were collected at 24, 48 and 72 hr, aliquoted and stored at −80°. IL-2 production was tested by enzyme-linked immunosorbent assay (ELISA). *CD14+ versus CD14−: P < 0·001 at 48 hr, P < 0·01 at 72 hr, not significant at 24 hr; †CD14+ versus CD14−/CD14CK: P < 0·05 at 48 and 72 hr, not significant at 24 hr. ‡CD14− versus CD14−/CD14CK: P < 0·05 at 48 and 72 hr, not significant at 24 hr; n = 8, anova. Bars show the mean value±1 SE. (Panels (c), (d) and (e) are on the following page)

Article Snippet: IL-1β, TNF-α and IL-6 depletion assays IL-1β (catalogue no. DLB50; R & D Systems, Minneapolis, MN), TNF-α (catalogue no. DTA50; R & D Systems) and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (catalogue no. D6050; R & D Systems) were used.

Techniques: Activation Assay, Incubation, Gentle, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Concentration Assay, Staining

Journal:

Article Title: Characterizing a soluble survival signal for activated lymphocytes from CD14 + cells

doi: 10.1046/j.1365-2567.2002.01463.x

Figure Lengend Snippet: A role of interleukin-6 (IL-6) in acting as the survival signal in the CD14 cocktails. (a) IL-6 in the CD14 cocktails was depleted by panning, as described in the Materials and methods. The original CD14 cocktails and the CD14 cocktails depleted of IL-6 by panning followed by blocking with anti-IL-6 monoclonal antibody (mAb) were used for the protection assay. *CD14− versus CD14−/CD14CK/IL-6D: P < 0·01 at 72 hr, not significant at 24 and 48 hr. n = 5, analysis of variance (anova). Bars show the mean value±1 SE. (b) CD14+ and CD14− peripheral blood mononuclear cells (PBMC) were prepared as described in the Materials and methods. To CD14− PBMC was added human recombinant IL-6 at concentrations of 0·5, 1, 2 and 4 ng/ml. CD14+ PBMC, CD14− PBMC and CD14− PBMC with different concentrations of IL-6 were stimulated with phytohaemagglutinin-p (PHA-p) at 37° in 5% CO2. At 24, 48 and 72 hr, the activation-induced cell death (AICD) was analysed by flow cytometry using propidium iodide (PI)/annexin staining. *CD14+ versus CD14−: P < 0·05 at 24, 48 and 72 hr; †CD14+ versus CD14−/IL-6/0·5 ng, CD14−/IL-6/1 ng, CD14−/IL-6/2 ng and CD14−/IL-6/4 ng: P < 0·05 at 24 and 48 hr, not significant at 72 hr; ‡CD14− versus CD14−/IL-6/1 ng, CD14−/IL-6/2 ng and CD14−/IL-6/4 ng: P < 0·05 at 72 hr, not significant at 24 and 48 hr; n = 5, anova. Bars show the mean value±1 SE. (c) To CD14− PBMC were added human recombinant IL-6 at concentrations of 0·5, 1, 2 and 4 ng/ml. CD14+ PBMC, CD14− PBMC and CD14− PBMC with different concentrations of IL-6 were stimulated with PHA-p at 37° in 5% CO2. At 24, 48 and 72 hr, the viable lymphocytes were counted in each well using the viable excluding dye Trypan blue. *CD14+ versus CD14−: P < 0·05 at 24, 48 and 72 hr; †CD14+ versus CD14−/IL-6 at 72 hr, P < 0·05 at all IL-6 concentrations except 4 ng/ml. ‡CD14− versus CD14−/IL-6 at 72 hr, P < 0·01 at an IL-6 concentration of 4 ng/ml, it is not significant at other, lower, IL-6 concentrations. n = 4, anova. Bars show the mean value±1 SE. (d) IL-6 was added into CD14− PBMC at concentrations of 0·5, 1, 2 and 4 ng/ml. CD14+ PBMC, CD14− PBMC and CD14− PBMC with IL-6 at different concentrations were cultured at 37° in 5% CO2. At 24, 48 and 72 hr time-points, the supernatants were collected and tested for IL-2 concentrations by enzyme-linked immunosorbent assay (ELISA). The data represents the mean of three independent experiments. *CD14+ versus CD14−: P < 0·0001 at 48 and 72 hr; it is not significant at 24 hr; †CD14+ versus CD14−/IL-6: P < 0·01 at 48 and 72 hr time-points at all IL-6 concentrations; it is not significant at 24 hr. ‡CD14− versus CD14−/IL-6: P < 0·05 at 72 hr at IL-6 concentrations of 2 and 4 ng/ml, it is not significant at other time-points and other, lower, IL-6 concentrations. n = 3, anova.

Article Snippet: IL-1β, TNF-α and IL-6 depletion assays IL-1β (catalogue no. DLB50; R & D Systems, Minneapolis, MN), TNF-α (catalogue no. DTA50; R & D Systems) and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (catalogue no. D6050; R & D Systems) were used.

Techniques: Blocking Assay, Recombinant, Activation Assay, Flow Cytometry, Staining, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay