Neutrophil differentiated HL-60 cells model Mac-1 (CD11b/CD18)-independent neutrophil transepithelial migration

Abstract

During active intestinal inflammation granulocytes accumulate in the lumen of the gut where they damage the epithelium through the release of various products such as reactive oxygen species and proteolytic enzymes. Previously, using function blocking monoclonal antibodies, we showed that neutrophil migration across intestinal epithelial monolayers in response to various chemoattractants was partially beta(2) integrin Mac-1 (CD11b/CD18)-independent. Here, we show that treating neutrophils with intact monoclonal antibody (mAb) to CD18 activates the cells to express more CD11b. Thus our goal now was to determine whether neutrophil Mac-1-independent transepithelial migration proceeds independently of prior cell activation through Mac-1. We took two approaches, one using blocking Fab' fragments of mAb to CD18 and the second was to develop a neutrophil differentiated HL-60 cell line which is Mac-1 deficient to further study neutrophil/epithelial cell interaction. Anti-CD18 Fab' minimally activated neutrophils but inhibited approximately 75% of transepithelial migration to fMLP while having a minimal effect (</=25% inhibition) on the migration to C5a. Upon incubation with dimethylsulphoxide, HL-60 cells differentiated and up-regulated CD11b expression and migrated to C5a and n-formyl methionyl leucyl phenylalanine in a similar manner to peripheral blood neutrophils. In contrast, CD11b expression was minimal on HL-60 cells differentiated with dibutytyl cAMP to a neutrophil-like phenotype. These cells, however, readily migrated across both intestinal and lung epithelial monolayers in response to C5a. We conclude that Mac-1-independent transepithelial migration does not require prior activation of cells via Mac-1 ligation because HL-60 cells lacking Mac-1 (CD11b/CD18) expression migrate effectively. HL-60 cells differentiated with dbcAMP should greatly assist in the search for the Mac-1-independent ligands for neutrophil migration across epithelium.

Figure 1

Effect of anti-β₂ integrin antibody on neutrophil migration and activation. (a) Effect of intact anti-β₂ integrin antibody on neutrophil migration across bare filters and T84 monolayers. Freshly isolated neutrophils were induced to migrate across bare filters or inverted T84 monolayers. Black bars: migration without added mAb, grey bars: migration in the presence of 30 µg/ml anti-β₂ antibody. Migration across T84 monolayers in the absence of chemoattractant was routinely less than 2%. The figure shows a representative of over five experiments across bare filters and over 20 experiments across T84 monolayers. Each bar is the mean of migration from three wells ± SD. Insert: Neutrophil migration across bare filters and T84 monolayers was assessed in the presence of binding isotype control anti-MHC class I antibody W6/32 (IgG2a). Black bars: migration without added mAb, grey bars: migration in the presence of W6/32 antibody. Each bar is the mean of migration from three wells ± SD. (b) Effect of intact anti-β₂ integrin antibody on neutrophil Mac-1 expression. Neutrophils were treated with anti-β₂integrin mAb or Fab′ fragments for 20 min at room temperature or left untreated. Bars indicate percentage increase in CD11b mean fluorescent intensity following intact antibody (n = 4) or Fab′ (n = 2) treatment of neutrophils relative to no anti-β₂ integrin mAb or Fab′ control. Bars represent the mean ± SD. Both IB4 and 60.3 anti-β₂ intact mAb were tested with similar results. (c) Effect of Fab′ fragments of anti-β₂ integrin mAb on neutrophil migration across bare filters and T84 monolayers. Migration was performed as in (a), except that Fab′ fragments were used instead of intact antibody. This experiment was repeated twice with similar results. Each bar is the mean of migration from three wells ± SD.

Figure 2

Mac-1 expression on neutrophils and HL-60 cells. (a) Mac-1 expression on freshly isolated neutrophils. Purified neutrophils were treated with chemoattractants for 30 min or left untreated (NT), then stained with anti-CD11b or anti-CD18 antibody. The mean fluorescence intensity is shown in the top-right corner of each histogram. One representative experiment is shown of 10 for CD11b and two for CD18. (b) Mac-1 expression on undifferentiated HL-60 cells. HL-60 cells were stained for CD11b or CD18 expression. One representative experiment of four for CD11b and three for CD18 is shown. (c) Mac-1 expression on the surface of DMSO-differentiated HL-60 cells. Cells were differentiated for 5 days with 1·2% DMSO, and then stained for CD11b and CD18. One representative experiment of three is shown. (d) Mac-1 expression on the surface of dbcAMP-differentiated HL-60 cells. Cells were treated with 500 µm dbcAMP for 2 days, washed and stained for CD11b and CD18. One representative experiment of four for CD11b and three for CD18 is shown.

Figure 3

Migration of granulocyte-differentiated HL-60 cells across bare filters and T84 monolayers. (a) Migration of DMSO-differentiated HL-60 cells. HL-60 cells were differentiated with 1·2% DMSO for 5 days. After washing and ⁵¹Cr labelling, 10⁵ HL-60 cells per filter were used in the migration assay. Each bar is the mean of percentage migration ± standard error of the mean (SEM) of three independent experiments. (b) Migration of dbcAMP-differentiated HL-60 cells. HL-60 cells were differentiated with 500 µm dbcAMP for 2 days, washed, labelled with ⁵¹Cr and used in the migration assay as described earlier. Each bar is the mean percentage migration of three independent experiments ± SEM.

Figure 4

Granulocyte-differentiated HL-60 cells migrate across T84 inverted monolayers. (a) DMSO-differentiated HL-60 cells migrate across T84 monolayers similar to neutrophils. DMSO dHL-60 cells were allowed to migrate across inverted T84 monolayers for 2 hr in response to 10⁻⁸ m C5a or 10⁻⁷ m fMLP. Black bars: migration to chemoattractant alone, open bars: migration to the chemoattractant in the presence of 30 µg/ml of intact anti-β₂ antibody. Each bar is the mean percentage migration of three independent experiments ± SEM. (b) β₂ integrin independent migration of dbcAMP-differentiated HL-60 cells in response to C5a. DbcAMP dHL-60 cells were stimulated, with the indicated concentrations of C5a, to migrate across inverted T84 monolayers for 2 hr. Bars are the mean percentage migration ± SEM from five experiments using dbcAMP-differentiated HL-60 cells and 10⁻⁸ m C5a as a chemoattractant across T84 monolayers (P = 0·1482). (c) Migration across T84 monolayers does not induce significant Mac-1 up-regulation on dbcAMP-differentiated HL-60 cells. HL-60 cells were differentiated for 2 days, washed and allowed to migrate in response to C5a for 2 hr across inverted T84 monolayers. Transmigrated cells were collected and stained for CD11b expression. Similar results were obtained in three experiments.

Figure 5

Granulocyte-differentiated HL-60 cells migrate across inverted A549 lung epithelial cell monolayers. The migration of dbcAMP dHL-60 cells across A549 human lung adenocarcinoma cells was measured as in Fig. 4(b) using C5a (2 × 10⁻⁹) as stimulus in absence or presence of anti-β₂ integrin mAb (30 µg/ml). Bars are the mean percentage migration ± SEM from three consecutive experiments (P = 0·1399).