Fibronectin augments monocyte adhesion to low-density lipoprotein–stimulated mesangial cells  Ravinder S. Chana, David C. Wheeler  Kidney International  Volume 55, Issue 1, Pages 179-188 (January 1999) DOI: 10.1046/j.1523-1755.1999.00250.x Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 1 Adhesion of U-937 monocytes to human mesangial cells. U-937 monocytes were coincubated with growth-arrested (A) or low density lipoprotein (LDL)-stimulated mesangial cells (B) in serum free medium for one hour. After washing, adhered monocytes were fixed and stained with crystal violet. Note the lack of adhesion to the base of the culture dish between mesangial cells (original magnification ×100). Kidney International 1999 55, 179-188DOI: (10.1046/j.1523-1755.1999.00250.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 2 Time-dependency of adhesion of U-937 monocytes to prestimulated human mesangial cells. Monocytes were incubated for one hour with mesangial cells that had been prestimulated for various time periods with low density lipoprotein (LDL; 100 μg/ml), minimally modified (MM)-LDL (100 μg/ml) and tumor necrosis factor α (TNFα; 100 U/ml). Adhered monocytes were fixed, stained, solubilized and quantitated by recording absorbance readings at 595nm. The mean (SEM) of triplicate absorbance readings, corrected for background staining of mesangial cells is expressed as percentage of control (unstimulated, growth arrested cells in RPMI). The results are representative of those obtained in 2 similar experiments. P vs. control: *P < 0.005, **P < 0.001 (ANOVA). Symbols are: (□) LDL; () MM-LDL; (▪) TNFα. Kidney International 1999 55, 179-188DOI: (10.1046/j.1523-1755.1999.00250.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 3 Adhesion of U-937 monocytes to human mesangial cells prestimulated with varying concentrations of LDL, MM-LDL and TNFα for 24 hours. (See Figure 2.) Monocytes were incubated for 1 hour with mesangial cells that had been prestimulated with various concentrations of LDL, MM-LDL and TNFα. The results shown are mean (SEM) of 6 experiments, each conducted in triplicate. P vs. control: *P < 0.05, **P < 0.005. Kidney International 1999 55, 179-188DOI: (10.1046/j.1523-1755.1999.00250.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 4 ICAM-1 expression by lipoprotein-stimulated cells. Growth arrested mesangial cells were exposed to various concentrations of LDL and MM-LDL for 24 hours. Expression of ICAM-1 was assessed by ELISA. The absorbance readings obtained (mean ± SEM) are expressed as percentage of control (unstimulated) cells (100%). Results are representative of 5 experiments, each conducted in quintuplicate (*P < 0.01 vs. control). Symbols are: () LDL; (▪) MM-LDL. Kidney International 1999 55, 179-188DOI: (10.1046/j.1523-1755.1999.00250.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 5 Time course of ICAM-1 expression. Growth arrested mesangial cells were exposed to LDL (100 μg/ml; ), MM-LDL (100 μg/ml; ) or TNFα (100 U/ml; ) for 4, 8, 24 and 48 hours. Results are expressed as mean (SEM) percentage over control (unstimulated) cells and are representative of 3 similar experiments, each conducted in quintuplicate. P vs. control: *P < 0.001, **P < 0.0001 (ANOVA). Kidney International 1999 55, 179-188DOI: (10.1046/j.1523-1755.1999.00250.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 6 VCAM-1 expression by lipoprotein-stimulated cells. (See Figure 4.) Growth arrested mesangial cells were exposed to LDL () and MM-LDL (▪) for 24 hours and VCAM-1 expression assessed by ELISA. The absorbance readings (mean ± SEM) obtained from stimulated cells are expressed as percentage of control (100%). Results are representative of 4 experiments, each conducted in quintuplicate. *P < 0.05 vs. control. Kidney International 1999 55, 179-188DOI: (10.1046/j.1523-1755.1999.00250.x) Copyright © 1999 International Society of Nephrology Terms and Conditions

Figure 7 Inhibition of monocyte binding to mesangial cells prestimulated with TNFα (A), MM-LDL (B), LDL (C), or unstimulated (D). Human mesangial cells were rested or prestimulated with TNFα (100 U/ml), MM-LDL (100 μg/ml) or LDL (100 μg/ml) for 24 hours and then incubated with anti-human ICAM-1 (CD54; 10 μg/ml), anti-human VCAM-1 (CD106; 4 μg/ml) and anti-human fibronectin antibodies (25 μg/ml) for between 2 and 3 hours. A suspension of U-937 monocytes was then added. Alternatively monocytes were incubated with anti-LFA-1 (20 μg/ml), anti-VLA-4(10 μg/ml) and anti-VLA-5 (5 μg/ml) antibodies for 30 minutes before being adding to rested or prestimulated mesangial cells for one hour. After washing, adherent monocytes were fixed, stained with crystal violet and solubilized in triton X-100. Absorbance readings were obtained at 595nm. The results are expressed as mean (SEM) absorbance, corrected for the background staining of mesangial cells, as a percentage of control (100%). These results are representative of 3 to 6 experiments, each conducted in triplicate at optimal antibody concentrations. P vs. control: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Kidney International 1999 55, 179-188DOI: (10.1046/j.1523-1755.1999.00250.x) Copyright © 1999 International Society of Nephrology Terms and Conditions