Figure 1.
Light microscopy and fluorescence microscopy images of non-differentiated and differentiated THP-1 cells.
Non-differentiated THP-1 cells (A) display a round shape and a nonadherent pattern while differentiated THP-1 cells (B) display a dendritic shape and an adherent pattern at light microscopy (×400), black scale bar : 10 µm. Fluorescence microscopy images (×200) showing CD11c expression by non-differentiated THP-1 cells (C) and differentiated THP-1 cells (D). Nuclei are stained in blue (DAPI) and CD11c is stained in red phycoerythrin (PE), white scale bar : 20 µm. A significantly increased CD11c expression was observed on differentiated THP-1 cells.
Figure 2.
Quantification of fluorescence intensity by non-differentiated and differentiated THP-1 cells.
Membrane expression of CD54 (P = 0.033, *), CD11b (P = 0.031, §) and CD11c (P = 0.002, #) was significantly increased by differentiated ▪ THP-1 cells compared to non-differentiated □ THP-1 cells. A significant decrease of CD33 expression (P = 0.049, +) were observed and no significant difference of CD86. Errors bars represent standard deviation.
Table 1.
Percentage of cytotoxicity of PMA differentiated THP-1 exposed to reagents.
Figure 3.
Flow cytometry analysis of phenotypic changes of macrophages after various stimulatory substances.
A: expression of CD11b : the expression of CD11b increased after benzalkonium chloride (BAK) stimulation (P = 0.033, *) as compared to phosphate buffered saline (PBS). B: expression of CD11c : the expression of CD11c increased with BAK (P = 0.005, *), DNCB (P = 0.004, #), lipopolysaccharide (LPS) (P = 0.035, §) or tumor necrosis factor alpha (TNF-α) (P = 0.033, +) as compared to PBS. C: expression of CD54 : the expression increased under LPS stimulation (P = 0.007, *) as compared to PBS. D: expression of CD33 : a decreased expression of CD33 was observed when exposed to BAK (P = 0.003, *) as compared to PBS. Errors bars represent standard deviation.
Figure 4.
Fluorescence microscopy images of carboxylate microspheres in non-differentiated THP-1 cells and in macrophages.
An increased carboxylate microspheres (red) concentration is observed in macrophages (B) than in non-differentiated THP-1 cells (A). Nuclei are stained in blue (DAPI). Magnification used was ×400. Bar : 20 µm.
Figure 5.
Quantification of carboxylate-modified fluorescent microspheres in non-differentiated THP-1 cells and macrophages.
Macrophages had a higher level of phagocytosis (48.3±3%) (P<0.0001, +) as compared to non-differentiated THP-1 cells (5.1±0.6%). Increased phagocytosis was observed after 24-h exposure to benzalkonium chloride (BAK) (P = 0.015, *), lipopolysaccharide (LPS) (P<0.0001, #) or tumor necrosis factor alpha (TNF-α) (P = 0.03, §) as compared to macrophages exposed to phosphate buffered saline (PBS). Errors bars represent standard deviation.
Figure 6.
Evaluation of migration rates of non-differentiated THP-1 cells and PMA differentiated macrophages.
The migration rate was higher when macrophages were exposed to benzalkonium chloride (BAK) (16.2±2.1%, P = 0.001,*), lipopolysaccharide (LPS) (15.1%±4.7%, P = 0.004, §) or tumor necrosis factor alpha (TNF-α) (29.9±4.7%, P<0.0001, #) as compared to macrophages exposed to phosphate buffered saline (PBS). Migration was significantly higher when macrophages were exposed to TNF-α as compared to BAK (P = 0.004). Migration was measured in coculture with conjunctival cells except for column 2. Errors bars represent standard deviation.
Table 2.
Quantification of cytokines in supernatants from non differentiated and PMA-differentiated THP-1 cells.