Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors.

The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.

A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages.

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides -89 to -78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the -89/-78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.

The role of lymphotoxin in the IL-2-driven differentiation of human lymphokine-activated T-killer (T-LAK) cells in vitro.

Brief stimulation of human peripheral blood mononuclear cells with PHA and subsequent coculture with IL-2 results by 5 days in cultures of human lymphokine-activated killer (T-LAK) cells. While IL-2 drives the proliferation of these cells in vitro, their maturation into functional effector cells capable of cytokine secretion and cell cytokines depends on the presence of other cytokines. The role of LT in the differentiation and proliferation of human T-LAK cells in vitro was investigated. Higher levels of LT than TNF were secreted by T-LAK cells during the first 5 days of the primary culture, then secretion levels dropped sharply. Human T-LAK cells cultivated with anti-LT rabbit antisera showed a slight reduction in growth compared to normal rabbit serum controls. In contrast, phenotypic analysis by FACS showed a decrease in CD4+ and an increase in CD8+ populations of T-LAK cells in the treated cultures. Addition of LT from the beginning of the T-LAK cell culture resulted in an increase in CD4+ and a decrease in CD8+ cell populations at day 7. In addition, the cytolytic activity of non-MHC-restricted cytotoxicity and NK-like activity of anti-LT cultured T-LAK cells was also effected. These data indicated that LT may have a role in differentiation of IL-2 stimulated human T-LAK cells in vitro.

Host-tumor interaction in ovarian cancer. Spontaneous release of tumor necrosis factor and interleukin-1 inhibitors by purified cell populations from human ovarian carcinoma in vitro.

The biological activity of tumor necrosis factor (TNF alpha/beta) and interleukin-1 beta (IL-1 beta) can be blocked by soluble, naturally occurring molecules--TNF alpha/beta binding proteins (BP-55 and BP-75), derived from the extracellular portion of the 55- and 75-kDa TNF alpha/beta membrane receptors, and IL-1 receptor antagonist (IL-1ra), respectively. We examined the levels of these cytokines and their inhibitors in cell-free ascites of 18 patients with advanced ovarian carcinoma by ELISA. Levels of both TNF BP and IL-1ra dramatically exceeded those of TNF and IL-1; thus, it is unlikely that these cytokines are active in ascites from patients with this disease. We then elutriated solid tumor samples from three additional patients, yielding pure populations of tumor cells, macrophages, and lymphocytes. Cells were cultured for up to 48 hr and the spontaneous production of TNF, IL-1, and their inhibitors was measured by ELISA. Tumor cells and macrophages both released inhibitors for TNF and IL-1. Tumor cells released IL-1ra and BP-55, while macrophages released IL-1ra and BP-75. Kinetic studies showed that both tumor cells and macrophages produced an initial burst of TNF alpha and IL-1 beta which was overtaken within 48 hr by a sustained production of TNF BP and IL-1ra. Lymphocytes released no TNF alpha or TNF beta, which alone suggests that tumor associated lymphocytes are locally quiescent in vivo. TNF and IL-1 inhibitors originate from tumor cells and tumor associated macrophages and probably block TNF and IL-1 activity locally and regionally in ovarian carcinoma patients. Whether this phenomenon contributes to the pathogenesis of this disease remains to be determined.