An Improved in Vitro Assay to Quantitate Chemotaxis of Rat Peripheral Blood Large Granular Lymphocytes (LGL)

We have developed an improved method to study the directed migration, or chemotaxis, of rat peripheral blood large granular lymphocytes (LGL) in vitro. A modified Boyden chamber technique was used to measure chemotaxis of LGL through polycarbonate filters that had been coated with different basement membrane components. LGL were found to adhere to collagen types I and IV, laminin and fibronectin. However, only collagen type IV was not in itself chemotactic for LGL. Migrated cells could be identified both morphologically and phenotypically as LGL on collagen type IV-coated filters after incubation with a chemotactic stimulus. LGL were found to display chemotaxis to a number of different stimuli, including the classical chemoattractant agents N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, and complement fragments present in activated sera. However, the degree of response to these stimuli was much less than that of isolated peripheral blood neutrophils or monocytes. In contrast, all three cell types showed increased chemotaxis to the diacyl glycerol analog 1-oleoyl 2-acetyl glycerol (OAG), which induced a 4-14 fold stimulation of migration. Induction of chemotaxis of LGL by OAG was time and dose-dependent, as confirmed using checkerboard assays. In summary, we have developed a rapid, quantitative method to measure chemotaxis of LGL in vitro. This technique may now be utilized to identify naturally occurring chemoattractants for LGL and to study the intracellular and regulatory events associated with LGL migration.