Under a variety of conditions, alveolar macrophages can generate early response cytokines (TNF-α, IL-1), complement components, and chemotactic cytokines (chemokines). In the current studies, we determined the requirements for TNF-α and the complement activation product C5a in chemokine production in vitro and in vivo. Two rat CXC chemokines (macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC)) as well as three rat CC chemokines (MIP-1α, MIP-1β, and monocyte chemoattractant protein (MCP)-1) were investigated. Chemokine generation in vitro was studied in rat alveolar macrophages stimulated with IgG immune complexes in the absence or presence of Abs to TNF-α or C5a. The rat lung injury model induced by IgG immune complex deposition was employed for in vivo studies. Abs to TNF-α or C5a were administered intratracheally or iv, and effects on chemokine levels in bronchoalveolar lavage fluids were quantitated by ELISA. Both in vitro and in vivo studies demonstrated the requirements for TNF-α and C5a for full generation of CXC and CC chemokines. In vitro and in vivo blockade of TNF-α or C5a resulted in significantly reduced production of chemokines. Supernatant fluids from in vitro-stimulated macrophages revealed by Western blot analysis the presence of C5a/C5a desArg, indicating intrinsic generation of C5a/C5a desArg by alveolar macrophages and explaining the higher efficiency of intratracheal vs iv blockade of C5a in reducing chemokine production. These results underscore the central role of both TNF-α and C5a, which appear to function as autocrine activators to promote CXC and CC chemokine generation by alveolar macrophages.