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

Abstract

Summary 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 β2 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.

Effects of Simulated Microgravity on Functions of Neutrophil-like HL-60 Cells

“…Human promyelocytic leukemia HL-60 cells obtained from American Type Culture Collection (Manassas, VA) were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycinin 5 % carbon dioxide (CO 2 ) humidified air at 37 • C. The cells were induced to undergo differentiation to granulocytes by incubating them at a concentration of 5 × 10 5 cells/mL in the presence of 1.25 % DMSO for 5 days (Miura et al 2000;Carrigan et al 2005). For effects of simulated microgravity, RCCS developed by NASA was used (Savary et al 2001;Singh et al 2010).…”

“…RCCS has been widely used for elucidating the effects of microgravity on lymphocytes (Bai et al 2011), macrophages (Wang et al 2014(Wang et al , 2015 and endothelial cells (Versari et al 2007). To date, no data has been reported about PMNs cultured in RCCS, probably because PMNs isolated from whole blood exhibit a relatively short half-life (several hours) (Lefort et al 2009;Carrigan et al 2005) thereby precluding longterm culture. Alternatively, human promyelocytic leukemia cell line HL-60 has been widely used as a model for leukocytes to study their functions, since they can be differentiated into monocyte-like, eosinophil-like, or PMN-like cells using distinct inducing agents (Collins et al 1977;Newburger et al 1979;Miura et al 2000;Tsiftsoglou et al 2003;Carrigan et al 2005).…”

“…To date, no data has been reported about PMNs cultured in RCCS, probably because PMNs isolated from whole blood exhibit a relatively short half-life (several hours) (Lefort et al 2009;Carrigan et al 2005) thereby precluding longterm culture. Alternatively, human promyelocytic leukemia cell line HL-60 has been widely used as a model for leukocytes to study their functions, since they can be differentiated into monocyte-like, eosinophil-like, or PMN-like cells using distinct inducing agents (Collins et al 1977;Newburger et al 1979;Miura et al 2000;Tsiftsoglou et al 2003;Carrigan et al 2005). Here we applied the RCCS bioreactor to unravel the effects of simulated microgravity on both intact HL-60 cells (promyelocytes) and dimethyl sulfoxide (DMSO)-differentiated HL-60 cells (PMN-like cells).…”

Transepithelial Migration of Neutrophils

“…Despite the overwhelming dependence of neutrophil migration on the b 2 integrins, CD18-independent transmigration can occur in some situations (37)(38)(39). In the intestine, b 1 and b 3 integrins play no role in neutrophil transepithelial migration (15).…”

A Chemokine Receptor CXCR2 Macromolecular Complex Regulates Neutrophil Functions in Inflammatory Diseases

“…HL-60 cells were differentiated into the granulocyte lineage with 1.2% Me 2 SO in Iscove's modified Dulbecco's medium with 10% FBS for 5-7 days as described (33). The differentiated HL-60 (dHL-60) cells were transfected with pTriEx-4 vector encoding CXCR2 C-tail fragments (WT, PDZ motif mutation AAA, or PDZ motif deletion ⌬TTL) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.…”