The effects that the Streptococcus pneumoniae-derived, proinflammatory toxin, pneumolysin (8.37 and 41.75 ng/mL), has on the production of interleukin (IL)-8 and tumor-necrosis factor (TNF)-alpha by human neutrophils have been investigated in vitro. Total and extracellular IL-8 and TNF-alpha were assayed by enzyme-linked immunosorbent assay, and flow cytometry and colorimetric procedures were used to detect intracellular cytokine and cytokine messenger RNA, respectively. Treatment of neutrophils with pneumolysin either alone or in combination with the chemotactic tripeptide, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 microM), resulted in a time-dependent (maximal at 6 h) increase in synthesis and release of IL-8 but not of TNF-alpha, which was associated with increased expression of IL-8 messenger RNA transcripts and was abrogated by either cycloheximide (10 microg/mL) or depletion of Ca(2+) from the cell-suspending medium. These interactions between the toxin and neutrophils may contribute to the exaggerated pulmonary inflammatory responses caused by pneumolysin-producing strains of the pneumococcus.