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

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 β(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

Flow cytometric analysis with a fluorescent formyl peptide receptor ligand as a new method to study the pharmacological profile of the histamine H2-receptor

The histamine H2-receptor (H2R) is a Gs protein-coupled receptor. Its activation leads to increases in the second messenger adenosine-3',5'-cyclic monophosphate (cAMP). Presently, several systems are established to characterize the pharmacological profile of the H2R, mostly requiring radioactive material, animal models or human blood cells. This prompted us to establish a flow cytometric analysis with a fluorescently labeled formyl peptide receptor (FPR) ligand in order to investigate the H2R functionally and pharmacologically. First, we stimulated U937 promonocytes, which mature in a cAMPdependent fashion upon H2R activation, with histamine (HA) or selective H2R agonists and measured increases in cAMP concentrations by mass spectrometry. Next, indicative for the maturation of U937 promonocytes, we assessed the FPR expression upon incubation with HA or H2R agonists. FPR expression was measured either indirectly by formyl peptide-induced changes in intracellular calcium concentrations ([Ca2+]i) or directly with the fluorescein-labeled FPR ligand fNleLFNleYK-Fl. HA and H2R agonists concentration-dependently induced FPR expression and potencies and efficacies of fMLP-induced increases in [Ca2+]i and FPR density correlated linearly. Accordingly, flow cytometric analysis of FPR expression constitutes a simple, inexpensive, sensitive and reliable method to characterize the H2R pharmacologically. Furthermore, we evaluated FPR expression at the mRNA level. Generally, quantitative real-time polymerase chain reaction confirmed functional data. Additionally, our study supports the concept of functional selectivity of the H2R, since we observed dissociations in the efficacies of HA and H2R agonists in cAMP accumulation and FPR expression