TLR agonist-mediated effects on chemotaxis. (A) The ability of human neutrophils treated with TLR agonists to chemotax to IL-8 was measured in Neuro Probe chemotaxis chambers. Cells that had transmigrated through the filter were quantitated using a fluorescent plate reader. Microscopy and manual cell counts gave similar results. *P < .005, as determined by Student t test, compared with untreated cells responding to IL-8. Error bars indicate standard deviation of triplicate measurements. Data are representative of 3 similar experiments conducted on 2 donors. CXCR1 (B) and CXCR2 (C) expression on neutrophils was assessed by flow cytometry following 2 hours of stimulation with LPS, lipopeptide, and poly I:C. Isotype controls for treated cells were not significantly different from untreated cells. Mean fluorescence intensities (MFIs) for expression of CXCR1 (D) and CXCR2 (E) of neutrophils following 2 hours of stimulation with the indicated TLR stimulus, with or without 90 minutes of pretreatment with GM-CSF.