The regulation of macrophage phenotype by neutrophils was studied in the sc polyvinyl alcohol sponge wound model in mice made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more TNF-α, 168% more IL-6, and 61% less TGF-β1 than those from controls. Wound fluid IL-10 was not different between the two groups, and IL-4 was not detected. Intracellular TNF-α staining was greater in cells isolated from neutropenic wounds than in those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in Transwell cocultures of LPS-stimulated J774A. 1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages). Overnight cocultures accumulated 60% less TNF-α and IL-6 than cultures of J774A. 1 alone. The suppression of cytokine release was mediated by a soluble factor (s), because culture supernatants from wound cells inhibited TNF-α and IL-6 release from LPS-stimulated J774A. 1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-α release from LPS-stimulated J774A. 1 cells. Wound cell supernatants also suppressed TNF-α and superoxide release from murine peritoneal macrophages. The TNF-α inhibitory factor has a molecular mass< 3000 Da and is neither PGE 2 nor adenosine. The present findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage phenotype.