IL-1β–stimulated TEM consists of PECAM-1 and IL-8–dependent components that are separable and additive.

(A) Confluent RHP monolayers were treated with 5 nm IL-8 (abluminal) and either 1:200 negative control rabbit serum (NC) or anti–IL-8 serum was added to the abluminal (bottom) chamber at the start of the TM assay. PMNs were allowed to transmigrate for 4 hours. In this representative experiment, data represent the mean ± SEM from a minimum of 3 transwells for each condition. (B) Confluent RHP monolayers were treated for 24 hours with 10 U/mL IL-1β (luminal), washed, and 500 000 PMNs/well were applied to the luminal chamber. Anti–IL-8 serum (1:200) or negative control NI rabbit serum was added to either the luminal (top) or abluminal (bottom) chamber in conjunction with (luminal) anti–PECAM-1 (Houston) or anti–MHC-1 antibodies (100 μg/mL) as indicated (PMN aliquots were treated identically to luminal conditions 20 minutes prior to addition to wells). PMNs were allowed to transmigrate for 4 hours. Transmigration rates are expressed as the proportion of PMNs migrating through the transwell filter compared with the total number of PMNs added at the start of the experiment. Column 1, “unblocked” TM (anti–MHC-1) in the setting of abluminal NI serum; Column 2, “unblocked” TM (anti–MHC-1) in the setting of abluminal anti–IL-8 serum; Column 3, “blocked” TM (Houston) in the setting of abluminal NI serum; Column 4, “blocked” TM (Houston) in the setting of abluminal anti–IL-8 serum; Column 5, “unblocked” TM (anti–MHC-1) in the setting of luminal NI serum; Column 6, “unblocked” TM (anti–MHC-1) in the setting of luminal anti–IL-8 serum. Data represent the mean ± SEM from a minimum of 3 transwells for each condition from 3 separate experiments. (*Columns 2, 3, and 4 are significantly different [P < .05] from column 1. **Column 4 is significantly different from columns 2 and 3 [P < .05].)