Studies of human granulocyte phagocytosis stimulation by tuftsin

Tuftsin (Thr-Lys-Pro-Arg), a natural immunomodulating peptide originally found to stimulate phagocytosis by polymorphonuclear leukocytes (PMNs), is now known to bind to both PMNs and monocyte-macrophages, affecting their phagocytosis and other functions. The potential roles of tuftsin in surgery-related infections have been documented using animal models. However, there have been some difficulties in demonstrating the phagocytosis-stimulating activity of tuftsin. In view of this, we have developed a suitable human PMN phagocytosis assay for tuftsin and performed preliminary kinetic studies. The assay was performed on 24-well plates between PMNs and fluorescent microspheres. The greatest effect of tuftsin over the control was observed under the following conditions: 15 min incubation at 37 degrees C with 5 micrograms/ml tuftsin and a 50:1 ratio of particle to PMN. Particles bound on the surface of PMNs were removed by washing and trypsin treatment, followed by centrifugation through fetal bovine serum. This allowed us to utilize flow cytometry in this study. A flow cytometric procedure was then successfully adapted to human PMN phagocytosis that established a high correlation between microscopic evaluation and flow cytometry of phagocytosis. In addition to the above determinations of the percentage of phagocytic cells, we evaluated the effect of tuftsin on the number of particles engulfed by PMNs. Under the above optimum conditions, tuftsin has greater impact on the number of particles engulfed than on the percentage of phagocytic cells.