CYTOKINE REGULATION ON THE SYNTHESIS OF NITRIC-OXIDE IN-VIVO BY CHRONICALLY INFECTED HUMAN POLYMORPHONUCLEAR LEUKOCYTES

To determine if nitric oxide (NO) is produced by chronically infected human polymorphonuclear leucocytes (PMNs) in vivo, inflamed exudates ( periapical exudates: PE) collected from periapical periodontitis patie nts were examined. Cell-free supernatants and cells were separated by centrifugation. Significant levels of nitrite concentrations were obse rved in the supernatants. The production of inducible NO synthase (iNO S) in highly purified PMNs derived from PEs was then immunocytochemica lly determined using rabbit anti-human iNOS antiserum. In vitro, human peripheral blood PMNs (PB-PMNs) isolated from patients were cultured with a combination of Esherichia coli-lipopolysaccharide (LPS), recomb inant human interferon-gamma (rhIFN-gamma) and/or interleukin-1 beta ( rhIL-1 beta). The stimulated PB-PMNs showed steady-state levels of nit rite. The stimulation of LPS, rhIFN-gamma and rhIL-1 beta showed more NO induction than that of LPS with either IFN-gamma or IL-1 beta, sugg esting the synergistic effects of cytokines. Cryostat sections of surg ically removed periapical tissues were also immunohistochemically exam ined for iNOS, IFN-gamma and IL-1 beta. Two-colour immunohistochemistr y revealed the interaction of iNOS-producing PMNs and IFN-gamma- or IL -1 beta-producing mononuclear cells. On the basis of these data, we co ncluded that with the stimulation of inflammatory cytokines derived fr om mononuclear cells, PMNs can spontaneously produce NO at the site of chronic infection. The present studies are consistent with a hypothes is suggesting that PMNs could be regulated and delicately balanced to produce NO by mononuclear cell-derived cytokines in vivo. NO-producing cells may play a pivotal role in chronic inflammation.