Evidence for a high-molecular-weight form of pig intestinal calcium-binding protein.

features of chronic periodontal disease and it has been demonstrated (Goodson et al., 1974; El Attar, 1976) that inflamed gingivae contain much higher concentrations of prostaglandin E, than normal tissue. Chronically inflamed gingivae are infiltrated with large number of macrophages, lymphocytes and plasma cells, which interact in the immune response. Products of these cell interactions may modulate the function of gingival cells. The aim of this study was to determine the effect of soluble mononuclear cell factors from peripheral blood on prostaglandin E production by cells derived from human gingiva. Heparinized peripheral venous blood was obtained from patients with periodontal disease and from healthy volunteers. The mononuclear cells were isolated using Histopaque (Sigma) and cultured in Dulbecco’s Modified Eagle’s medium, supplemented with 10% foetal calf serum at 37OC in an atmosphere of CO,/air (1 : 19, v/v) in air. After a 72h incubation period, medium was removed, centrifuged and this cell-free supernatant medium was used as the source of mononuclear cell factors. Inflamed gingival tissue was obtained from patients undergoing routine periodontal surgery. Normal healthy gingival tissue was obtained from children undergoing surgery before orthodontic treatment. Fibroblast-like cells were obtained either from explant cultures or by enzymic (Dispase, Boehringer) digestion of the tissue. Cells were cultured in Minimal Essential medium containing 10% foetal calf serum. All mononuclear cell cultures tested released a factor that stimulated prostaglandin E production (as measured by radioimmunoassay) and all cell lines, whether derived from inflamed or normal gingivae, were responsive to the factor. Stimulation occurred in a dose-dependent manner, a 1 in 5 dilution of mononuclear-cell-factor-containing medium giving a several hundred-fold increase in prostaglandin E production over a 72 h incubation period. A dose-dependent stimulation was also observed when fragments of gingiva were incubated with mononuclear cell factors. All stimulatory effects were inhibited when incubations were carried out in the presence of indomethacin ( 1 4 p ~ ) , an inhibitor of prostaglandin biosynthesis. Mononuclear-cell-factor stimulation of prostaglandin synthesis was first observed after a 2 h exposure and this was markedly inhibited by cycloheximide (1 pg ml-I), suggesting that stimulation is dependent on synthesis of protein de nouo. However, this incubation was less marked on longer (up to 4 h) incubation with cycloheximide, which may have been due to metabolism of this compound. Brief exposure (0.5-2 h) to mononuclear cell factor did not result in stimulation of prostaglandin E synthesis during a subsequent 72 h incubation in the absence of mononuclear cell factor. Addition of arachidonate, the substrate for prostaglandin biosynthesis, at 1-15,u~, in the presence of mononuclear cell factors (1 in 25 dilution) resulted in a further dose-dependent stimulation of prostaglandin E production, whereas arachidonate produced no stimulation of basal prostaglandin E production. The responsiveness of the gingival cell lines to mononuclear cell factors decreased quite markedly with passage of the lines, perhaps due to loss of cell-surface receptors. The factor is heat-labile, losing 83% of its activity after treatment at 56OC for 30min and it is resistant to trypsin digestion (30min at 37OC). In the view of these findings it is possible that factors such as mononuclear cell factors may play some role in the complex pathogenesis of different chronic inflammatory diseases.