2004. much less TNF- and MIP-3 in time course and dose-response experiments. No MyD88-dependent or -independent response to endotoxin was seen in TLR4-deficient cell lines (C3H/HeJ and 23ScCr) and response was restored in TLR4-MD-2-transfected human embryonic kidney 293 cells. Blocking the MyD88-dependent pathway by DNMyD88 resulted in significant reduction of TNF- release but did not influence nitric oxide release. IFN- polyclonal antibody and IFN-/ receptor 1 antibody significantly reduced nitric oxide release. endotoxin was a potent agonist of both the MyD88-dependent and -independent signaling pathways of the TLR4 receptor complex of human macrophages. 55:B5 and LPS, at the same picomolar lipid A concentrations, selectively induced the MyD88-dependent pathway, while LPS activated the MyD88-independent pathway. Bacterial endotoxins of human pathogens interact with macrophages and other cells via the Toll-like receptor 4 (TLR4)-MD-2 receptor complex (TLR4 complex) (13), resulting in cellular activation, the release of cytokines, chemokines, reactive oxygen species, nitric oxide (4) and, in some individuals, the clinical picture of sepsis. However, other endotoxins often found in commensal species act as antagonists of the TLR4 complex (14, 44). Two signaling pathways have been described following TLR4 activation, the MyD88-dependent (9, 26) and -independent pathways (15, 33). These signaling pathways depend on Toll/interleukin-1 receptor adaptor proteins including MyD88, Mal/TIRAP (10, 17), TRIF/TICAM-1 (32, 47), and TRAM/TICAM-2 (11, 48). Endotoxin activation of the MyD88-dependent pathway results in rapid NF-B activation and release of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-), interleukin (IL-)1, IL-6, and chemokines like monocyte chemoattractant protein 1 (MCP-1), macrophage inflammatory protein 3 (MIP-3), and IL-8. Endotoxin activation of the MyD88-independent pathway results in rapid activation of interferon regulatory factor 3 (IRF3) (22, 33) leading to beta interferon (IFN-) release but delayed NF-B activation (15). IFN- binds to the type 1 IFN-/ receptor, which activates STAT1 (1, 23, 36) and leads to type 1 IFN-/, IFN–inducible protein 10 (IP-10), MCP-5, RANTES, and nitric oxide release (22, 35, 46). Further, TLR4 activation by endotoxin via the MyD88-independent (TRIF/TICAM-1) pathway is important for dendritic cell maturation and provides an important link between the innate and adaptive immune responses (16). How endotoxin interacts with the TLR4 complex molecules and recruits specific adaptor proteins remains poorly understood. Lipid A structure and configuration of endotoxin are known to determine the agonist or antagonist activity (24, 27, 37). For example, the KDO2-lipid A structure of meningococcal lipooligosaccharide (LOS) is TRAM-34 necessary for the maximal agonist activation of macrophages via the TLR4 complex (51). The aim of the study was to further determine the influence of endotoxin structure on TRAM-34 activation of the TLR4-MyD88-dependent and -independent signaling pathways. MATERIALS TRAM-34 AND METHODS Reagents. RPMI 1640 medium, Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin/streptomycin, sodium pyruvate, and nonessential amino acids were obtained from Cellgro Mediatech (Herndon, VA). Phorbol myristate acetate was purchased from GibcoBRL (Grand Island, NY). Human and mouse TNF-, IFN- enzyme-linked immunosorbent assay (ELISA) kits were from R&D systems (Minneapolis, MN). The RAW 264 and 7, 23ScCr (TLR4-deficient) cell lines were purchased from the American CTSL1 Type Culture Collection. TRAM-34 The THP-1 cell line was provided by Fred Quinn (Centers for Disease Control and Prevention, Atlanta, GA). The U937 cell line was obtained from Yusof Abu Kwaik (University of Kentucky School of Medicine, Lexington, KY). The MM6 cell line was provided by Geert-Jan Boon (Complex Carbohydrate Research Center, University of Georgia, Athens, GA). The C3H/HeJ (TLR4?/?) cell line was a gift from Bruce Beutler (Scripps Research Institute, La Jolla, CA). Anti-interferon-/ receptor 1 was purchased from Sigma (Saint Louis, Missouri). Rabbit anti-mouse IFN- and anti-mouse IFN- polyclonal antibodies were purchased from PBL Biomedical Laboratories via R&D Systems. LOS and LPS purification and quantitation. Endotoxin (lipooligosaccharide, LOS) from the serogroup B strain NMB (L2 immunotype) and strains 981 (L5 immunotype) and 44/76 (L3 immunotype) was extracted by the phenol-water method (34). Enteric lipopolysaccharide (LPS) (Sigma Chemicals, St. Louis, MO) used were 055:B5, 111:B4,.