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. 2016 Feb;186(2):297–311. doi: 10.1016/j.ajpath.2015.10.015

Figure 1.

Figure 1

Targeting of Lewis X (Lex) blocks polymorphonuclear leukocyte (PMN) transepithelial migration and increases PMN adhesive interactions. A: Epithelial cells were cultured to confluency on porous polycarbonate filters (Transwell). Human PMNs incubated with 10 μg/mL anti-Lex monoclonal antibodies (mAbs; H198, W6D3, and F8A1.1), isotype-matched control (Ctrl) mAbs (anti-IgG and anti-IgM), or anti-CD66b mAb as an anti-IgM binding control, were placed in the upper chamber of Transwell filters and induced to migrate into the 100-nm N-formyl-l-methionyl-leucyl-l-phenylalanine (fMLF)–containing lower Transwell chamber in the physiologically relevant basolateral-to-apical direction. Numbers of migrated PMNs were quantified by myeloperoxidase (MPO) assay, as described in Materials and Methods. Human PMNs incubated with 10 μg/mL anti-Lex mAbs (H198, W6D3, and F8A1.1) or isotype control mAbs (anti-IgG and anti-IgM) were added to the top of collagen-coated Transwell filters and induced to migrate into a lower chamber containing 100-nm fMLF (B) or 100-nm IL-8 (C). PMN chemotaxis was quantified by MPO assay (as described in Materials and Methods). D: Human PMNs incubated with 10 μg/mL anti-Lex mAbs (H198 or W6D3) or isotype control mAbs (anti-IgG and anti-IgM) were added to confluent human dermal microvascular endothelial cell (HDMEC) monolayers. PMNs were allowed to migrate for 1 hour in response to a 10 nmol/L gradient of fMLF. The number of migrated PMNs was quantified by MPO assay (as described in Materials and Methods). E: BCECF [2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester]–labeled PMNs were pretreated with 20 μg/mL H198, W6D3, CBRM1/29 (as a positive control for inhibition of PMN adhesion to T84 IECs), or isotype-matched control mAbs (anti-IgG or anti-IgM) before addition to confluent T84 intestinal epithelial cell monolayers. After 2 hours, nonadherent PMNs were removed by gentle washing, and adherent PMNs were lysed by addition of Triton-X. Adherence was measured by quantifying fluorescence at 485 nm. Data shown are fold change in fluorescence intensity for treatment (Tx) with anti-Lex mAbs (H198 or W6D3) or anti-CD11b/CD18 mAb versus isotype-matched control mAbs. Data depict means ± SEM (A–C and E). N = 5 independent donors (A); N = 3 independent donors (B–E). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.