Figure 5.

Role of the Fc portion of adalimumab in the regulation of CD36 expression. (a) Isolation of F(ab')2, the antigen binding fragment, from adalimumab with pepsin digestion. The purity of the F(ab')2 fragment obtained was verified by migration of the specimens obtained on a 12% denaturant acrylamide gel according to the manufacturer's instructions. Lane 1 represents the standard molecular weight (20 to 250 kDa). Lane 2 represents the light chain (25 kDa) and the heavy chain (50 kDa) of adalimumab. Lane 3 represents the intact light chain (25 kDa) and truncated heavy chain (30 kDa) obtained after pepsin digestion. (b) The F(ab')2 fragment of adalimumab increases membrane expression of CD36. Monocytes were incubated with macrophage-serum-free medium (M-SFM) alone (control), or with M-SFM containing the purified F(ab')2 fragment from adalimumab at an equimolar concentration to that of 1 μg/ml adalimumab (0.8 μg/ml of F(ab')2 fragment being equivalent to 1 μg/ml of adalimumab) for 24 h and membrane expression of CD36 in monocytes was quantified using flow cytometry. Data represent the geometric mean ± standard error of the fluorescence measured in three experiments in duplicate. *Significantly different from the control (p < 0.05). (c) The F(ab')2 fragment of adalimumab induces reactive oxygen species (ROS) production. Monocytes were incubated with Hanks balanced salt solution (HBSS) alone (control), or with HBSS containing F(ab')2 (0.8 μg/ml) for 1 h. ROS production was measured by chemiluminescence in the presence of 5-amino-2,3-dihydro-1,4-phthalazinedione in a thermostatically controlled luminometer. Data represent total chemiluminescence emission (area under the curve) for 1 h, measured in three experiments. *Significantly different from the control (p < 0.05).