The MultiScreen filtration system to measure chemoattractant-induced release of leukocyte granule enzymes by...

A variety of chemoattractants initiate chemotaxis by selective binding to chemoattractant receptors (CARs), a subfamily of seven transmembranous G-protein coupled receptors (7TM-GPCRs) expressed in the leukocyte plasma membrane. Whatever the chemoattractant, signaling leading to chemotaxis involves several common biological steps which occur within seconds to minutes of CAR ligand binding. Though each step can be used to study the progress of the chemotaxis activation process. only certain biological events are suitable for monitoring chemotaxis signaling on large sample numbers as required for drug screening. An example of such is the release of granule enzymes by leukocytes in response to a CAR ligand. In this study, promyelocytic HL-60 cells were employed to set up a 96-well microplate methodology using filtration instead of centrifugation to collect the extracellular fluid together with the cell-released enzymes. Undifferentiated HL-60 cells were found not to respond to any of the CAR ligands. With various types of HL-60 cells which had differentiated along the neutrophilic or monocytic pathways, a large enzyme release was dose-dependently triggered by fMLF or C5a, but none of the tested CC or CXC chemokines. The highest responsiveness was found for neutrophilic HL-60 cells differentiated with dibutyryl cyclic AMP. With normal human monocytes (prepared from the blood of healthy donors by leukapheresis and elutriation), the granule enzyme release response was large to fMLF or C5a, substantial to MCP-1, low to RANTES or MIP-1alpha, but insignificant to Eotaxin, IL-8 and GROalpha. The method readily measures N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase and elastase activities, and requires approximately five times fewer cells than classical methods, a very important feature when normal human cells are to be used in screening assays. The method was also adapted to large scale screening of antagonists such as cyclosporins A and H for fMLF-mediated signaling using HL-60 cells and monocytes, and truncated (9-76) MCP-1 for MCP-1-mediated signaling using monocytes.