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Production of nitric oxide (NO) by LPS-activated macrophages is due to a complex cellular signaling initiated by TLR4 that leads to the transcription of IFN-β, which activates IRF-1 and STAT-1, as well as to the activation of NF-κB, required for iNOS transcription. High concentrations of LPS can also be uptaken by scavenger receptors (SRs), which, in concert with TLR4, leads to inflammatory responses. The mechanisms by which TLR4 and SRs interact, and the pathways activated by this interaction in macrophages are not elucidated. Therefore, our main goal was to evaluate the role of SRs, particularly SR-A, in LPS-stimulated macrophages for NO production. We first showed that, surprisingly, LPS can induce the expression of iNOS and the production of NO in TLR4−/− mice, provided exogenous IFN-β is supplied. These results indicate that LPS stimulate receptors other than TLR4. The inhibition of SR-A using DSS or neutralizing antibody to SR-AI showed that SR-A is essential for the expression of iNOS and NO production in stimulation of TLR4 by LPS. The restoration of the ability to express iNOS and produce NO by addition of rIFN-β to inhibited SR-A cells indicated that the role of SR-AI in LPS-induced NO production is to provide IFN-β, probably by mediating the internalization of LPS/TLR4, and the differential inhibition by DSS and neutralizing antibody to SR-AI suggested that other SRs are also involved. Our results reinforce that TLR4 and SR-A act in concert in LPS activation and demonstrated that, for the production of NO, it does mainly by synthesizing IRF-3 and also by activating the TRIF/IRF-3 pathway for IFN-β production, essential for LPS-mediated transcription of iNOS. Consequently STAT-1 is activated, and IRF-1 is expressed, which together with NF-κB from TLR4/MyD88/TIRAP, induce iNOS synthesis and NO production. TLR4 and SRs act in concert activating IRF-3 to transcribe IFN-β and activate STAT-1 to produce NO by LPS-activated macrophages. • LPS-activated TLR4-deficient cells are able to produce NO provided IFN-β is supplied. • SRs are indispensable for TLR4-mediated NO production in macrophages. • SR-AI acts in concert with TLR4 to internalize LPS and activate TRIF/IRF-3 pathway. • SR-AI is needed for TLR4-mediated expression of IFN-β, crucial for iNOS expression. • SR-AI is needed for activation of STAT-1 and expression of IRF-1, crucial for iNOS expression. [ABSTRACT FROM AUTHOR]