Fig. 2.
HLA I IgG stimulation increased the transmigration of human monocytes. (A) ECs were seeded in the upper chamber on day −1 and grown to a confluent monolayer. On day 0, the EC were washed and starved for four hours, and then left unstimulated (UT) or stimulated with 1 μg/mL HLA I antibody (intact or F(ab’)2) or hIgG for an additional hour. For TNF-α stimulation, 10 ng/mL TNF-α was added at the beginning of the EC starvation. The unbound antibodies and TNF-α were extensively washed away before adding the monocytes. The cells were co-cultured for five days before phenotype or gene expression assessments. During the co-culture, half of the medium was replaced every two days, before which the supernatants were collected for cytokine detection. (B) Transmigrated mononuclear cells were imaged on day 2 before medium replacement and counted. Fold changes in mean numbers of transmigrated mononuclear cells co-cultured with unstimulated (UT), HLA IgG or HLA-I F(ab’)2 stimulated EC are shown by box and whiskers graph. Data were from two independent experiments using two monocyte donors cultured with ECs from three donors. *p < 0.05 was analyzed by repeated measures one-way ANOVA with Sidak’s tests.