In this study, we sought to determine if human polymorphonuclear leukocytes (PMNs) derived from chronically inflamed tissues can produce inflammatory cytokines in vivo. Human gingival crevicular fluid (GCF) with adult periodontitis was collected, and PMNs in GCF were examined after purified by Ficoll-Hypaque gradient method. Cytokines from peripheral blood (PB) cells stimulated with concanavalin A, LPS, or zymosan were also characterized, since GCF contains predominantly PMNs (> 95%) with a small number of lymphocytes or macrophages. Production of interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF alpha), and IL-6 in GCF or culture supernatants of peripheral blood cells was determined by ELISA. Significant levels of IL-1 alpha and IL-1 beta secretion were found in GCF. PB cells in culture showed prominent cytokine production from monocytes/macrophages, followed by lymphocytes. Human peripheral blood PMNs (PB-PMNs) also produced low levels of IL-1 alpha and IL-1 beta, but not TNF alpha and IL-6. These cells were also examined for cytokine mRNA expression using the reverse transcription-polymerase chain reaction analysis. Highly purified PMNs (> 99.5%) from GCF expressed mRNA for IL-1 alpha, IL-1 beta and TNF alpha, but not for IL-6. PB-PMNs in culture also showed mRNA expression for IL-1 alpha, IL-1 beta, and TNF alpha in a time- and dose-dependent manner, especially after stimulation with zymosan. Therefore, we concluded that human PMNs from inflamed tissues can produce IL-1 alpha, IL-1 beta, and TNF alpha in vivo, but not IL-6.