The complex of porcine seminal plasma heterodimers I and II (PSP-I/PSP-II), which are heterodimers of glycosylated spermadhesins, is the major component of porcine seminal fluid. The proinflammatory and immunostimulatory activities of this spermadhesin complex suggest its participation in modulation of the uterine immune activity that may ensure reproductive success. Spermadhesin PSP-I/PSP-II induced the migration of neutrophils into the peritoneal cavity of rats via activation of resident cells. In the present study, we have investigated the involvement of macrophages and mast cells in the neutrophil chemotactic activity of PSP-I/PSP-II and the underlying mechanism. Macrophages and mast cells were isolated, cultured, and stimulated with purified PSP-I/PSP-II. Pharmacological modulation was performed using the glucocorticoid dexamethasone, indomethacin (cyclooxygenase inhibitor), MK886 (leukotriene inhibitor), and the supernatant of spermadhesin-stimulated mast cells. Macrophages stimulated with PSP-I/PSP-II released into the culture supernatant a neutrophil chemotactic substance. This activity was partly inhibited by both dexamethasone (85%) and the supernatant of spermadhesin-stimulated mast cells (74%) but not by indomethacin and MK886. An anti-tumor necrosis factor (TNF) α antibody neutralized (by 68%) the neutrophil chemotactic activity of PSP-I/PSP-II-stimulated macrophages. An anti-interleukin (IL)-4 antibody blocked the inhibitory activity of spermadhesin-stimulated mast cells on release of a neutrophil chemotactic substance by PSP-I/PSP-II-stimulated macrophages. As a whole, these data indicate that the neutrophil migration-inducing ability of spermadhesin PSP-I/PSP-II involves the release of the inflammatory cytokine TNFα by stimulated macrophages and that this activity is modulated by the lymphokine IL-4 liberated by mast cells. The balance between these two cytokines may control onset of the local inflammatory reaction, avoiding excessive neutrophil recruitment that would lead to tissue damage.