Table 1.
Patient characteristics (n.a. = not applicable).
Figure 1.
Ex vivo assessment of phagocytosis.
A. After MACS separation monocytes were gated according to their properties in the forward and side scatter. B. Phagocytosis of non-opsonized fluorescent polystyrene-based latex beads. Each mean fluorescence is considered to be an equivalent of the total number of latex beads phagocytosed by 1×106 cells. All mean fluorescences are related to the background control (cells without beads). C. Monocytes from glatiramer acetate (GA) treated MS patients (n = 13) exhibit a significantly higher phagocytic activity than those of healthy donors (n = 10) or non-treated MS patients (n = 20). Significant effects are indicated by asterisks (*p<0.05 and **p<0.01 using Bonferroni's Multiple Comparison Test) as determined by one-way ANOVA.
Table 2.
Evaluation of cell viability by the alamar blue® (resazurin) dye assay after 24 h of treatment with glatiramer acetate (GA) or the mannitol vehicle control.
Table 3.
Average mean (± standard deviation) of the percentages of monocytes positive for either annexin V, PI or both obtained from gate properties of the FL1 and FL3 sensor.
Figure 2.
In vitro assessment of phagocytosis.
A–B, D. Data are expressed as means of the mean fluorescence intensities (MFI) ± SEM of three independent experiments. A–D. The MFI displays the amount of incorporated fluorescent latex particles phagocytosed by 3×105 cells. Significant effects vs. medium control(s) are indicated by asterisks (*p<0.05, **p<0.01, and ***p<0.001 using Bonferroni's Multiple Comparison Test) as determined by one-way ANOVA. A. Monocytes isolated from healthy donors were used to assess the dose and time course of glatiramer acetate (GA) on the ingestion of non-opsonized polystyrene-based latex particles. B. After 3 h of incubation low concentrations of GA significantly increased phagocytosis. C. Histogram shows the distinct shift of the MFI after 3 h of GA treatment. One representative experiment is shown. D. Adhesion of polystyrene beads did not have a relevant impact on the MFI as shown by cytochalasin D (50 µM) experiments.
Figure 3.
Visualization of phagocytosis.
A–D. Immunocytochemistry showed distinct phagocytosis of polystyrene beads (green) by monocytes treated with 31.25 µg/ml glatiramer acetate (GA) for 24 h (red, labelled with anti-CD14 antibody) as compared to medium, human serum albumin (HA), and mannitol (Mt) vehicle control. Scale bar: 50 µm. E. Representative z-stack series of confocal microscopic images demonstrate that the particles were completely internalised and not merely attached to the outer membrane. Scale bar: 10 µm.
Figure 4.
A–B. Gated monocytes in a forward vs. side scatter dot-plot analysis of peripheral blood mononuclear cells. Monocytes were then gated according to their surface expression of CD14 and CD16. Flow cytometric analysis of phagocytosis revealed that CD14++CD16+ monocytes (gate III) engulfed polystyrene beads more effectively than the other subsets (gate I and II). CI–III. The mean fluorescence intensities represent the amount of incorporated fluorescent latex particles phagocytosed by 3×105 cells. D. Increase in the percentage of CD14+CD16+ monocytes after 4 h and 8 h of treatment with 31.25 µg/ml glatiramer acetate (GA) in MACS isolated monocytes. Data are expressed as mean percentages ± SEM of three independent experiments. Significant effects vs. controls are indicated by asterisks (*p<0.05, **p<0.01, and ***p<0.001 using Bonferroni's Multiple Comparison Test) as determined by one-way ANOVA. E. Slight but not significant increase of CD16 expression after GA treatment.
Figure 5.
Exclusion of soluble factors led to the analysis of the expression of several phagocytic receptors.
A–B. The amount of incorporated fluorescent latex particles phagocytosed by 3×105 MACS isolated monocytes is displayed by the mean fluorescence intensities. Data are expressed as means of the mean fluorescence intensities (MFI) ± SEM of three independent experiments. A significant effect vs. medium control is indicated by asterisks (*p<0.05 and ***p<0.001 using Bonferroni's Multiple Comparison Test) as determined by one-way ANOVA. A. Monocytes were treated for 3 h with the denoted substances. In particular, IL-10 treatment did not increase phagocytosis. B. Treatment of freshly isolated monocytes for 6 h with conditioned supernatants from monocytes, which were previously treated for 12 h with GA, did not have an impact on phagocytosis. C. Surface expression of several receptors or subunits of such receptors involved in phagocytosis after a treatment period of 24 h.
Table 4.
Mean fluorescence intensities of CD36, CD210, and TIM-3 (± SEM) of three independent experiments after a treatment period of 24 h according to the different monocyte subsets, i.e. CD14+CD16− and CD14+CD16+ monocytes.
Figure 6.
GA decreases CD11c expression.
A. Significant decrease in CD11c expression after 24 h of GA treatment. B. 24 h treatment with 3.9 µg/ml and 31.25 µg/ml GA treatment dose-dependently decreased the percentage of phagocytosing monocytes (see M2 gate) and increased the percentage of non-phagocytosing monocytes (see M1 gate) of fibrinogen-coated beads. Data are expressed as mean percentages ± SEM of three independent experiments. Significant effects vs. GA 31.25 µg/ml or GA 3.9 µg/ml, respectively, are indicated by asterisks (*p<0.05, **p<0.01, and ***p<0.001 using Bonferroni's Multiple Comparison Test) as determined by one-way ANOVA.
Figure 7.
The phagocytosis of polystyrene beads was most significantly decreased by anti-CD36 antibodies.
The MFI displays the amount of incorporated beads phagocytosed by 3×105 cells. Data are expressed as means of the mean fluorescence intensities (MFI) ± SEM of three independent experiments. Pre-treatment of monocytes with anti-CD14, anti-CD16, anti-CD32, anti-CD210, and anti-TIM3 antibodies reduced glatiramer acetate (GA) induced phagocytosis of polystyrene beads. The anti-CD36 and, at the highest concentration, the anti-IL10 antibody most effectively suppressed phagocytosis. Significant effects vs. GA 31.25 µg/ml are indicated by asterisks (*p<0.05, **p<0.01, and ***p<0.001 using Bonferroni's Multiple Comparison Test) as determined by one-way ANOVA. B–C. The anti-CD36 antibodies of two different clones show a diverse impact on the phagocytosis of polystyrene beads by untreated cells, whereas both antibodies effectively decreased the GA induced phagocytosis.
Figure 8.
Anti-CD36 antibodies did not revert the GA mediated inhibition of OxLDL uptake.
A. Monocytes were gated within PBMC according to their light scatter characteristics. B. Glatiramer acetate (GA) and medium control without any preceding treatment within the marker M1 served as a control for the analysis of monocytes that did not ingest labelled low density lipoprotein (DiO-OxLDL). C. At the indicated timepoints GA treated cells exhibited a slight left-shifted fluorescence signal, which was most prominent after 3 h of DiO-OxLDL incubation. D. Data are expressed as the percentages of monocytes that did not ingest DiO-OxLDL. After 1 h and 3 h the percentages of those cells were significantly higher in GA treated cells than in untreated cells. E–F. The percentages of non-ingesting cells are higher at higher concentrations as compared to lower concentrations of GA suggesting a dose-depending effect. G. The anti-CD36 antibody from the clone FA6–152 distinctly increased the amount of non-ingesting cells, while another anti-CD36 antibody (clone 255606) did not have an impact.
Table 5.
Percentages of monocytes positive for either annexin V, PI or both obtained from gate properties of the FL1 and FL3 sensor treated with an anti-CD36 antibody, clone 255606, in low (0.1 µg/ml) and high (1 µg/ml) concentrations, with or without 31.25 µg/ml Glatiramer Acetate (GA).
Table 6.
Percentages of monocytes positive for either annexin V, PI or both obtained from gate properties of the FL1 and FL3 sensor treated with an anti-CD36 antibody, clone FA6–152, in low (0.1 µg/ml) and high (1 µg/ml) concentrations, with or without 31.25 µg/ml Glatiramer Acetate (GA).