Interferon-gamma and bacterial lipopolysaccharide act synergistically on human neutrophils enhancing interleukin-8, interleukin-1beta, tumor necrosis factor-alpha, and interleukin-12 p70 secretion and phagocytosis via upregulation of toll-like receptor 4.

In human neutrophils, interferon (IFN)-gamma enhanced the expression of toll-like receptor 4 (TLR4), a crucial component of the signaling receptor complex for bacterial lipopolysaccharide (LPS). Lipopolysaccharide alone did not affect TLR4 expression, but costimulation with IFN-gamma and LPS induced higher levels of TLR4 expression than stimulation with IFN-gamma alone. Using the protein synthesis inhibitor cycloheximide and measuring the expression of CD35 in neutrophils stimulated with IFN-gamma and LPS alone or in combination, we could demonstrate that IFN-gamma enhances TLR4 by de novo protein synthesis, whereas the addition of LPS acts synergistically by enhancing vesicular mobilization to the cell surface. Costimulation with IFN-gamma and LPS induced neutrophil activation and enhanced secretion of the cytokines, interleukin (IL)-8, IL-1beta, tumor necrosis factor-alpha, and IL-12 p70, and phagocytosis of latex beads, processes that were blocked by a monoclonal antibody specific for TLR4. These data suggest that IFN-gamma primes neutrophils to respond to LPS.

Moxifloxacin inhibits cytokine-induced MAP kinase and NF-kappaB activation as well as nitric oxide synthesis in a human respiratory epithelial cell line.

BACKGROUND: We previously demonstrated that the quinolone moxifloxacin prevents Candida albicans pneumonitis and epithelial nuclear factor kappaB (NF-kappaB) nuclear translocation in immunosuppressed mice. OBJECTIVES: To explore the anti-inflammatory effects of moxifloxacin directly on a lung epithelial cell line. METHODS: We studied the effect of clinically relevant concentrations of moxifloxacin (2.5-10 mg/L) on cytokine-induced activation of nitric oxide (NO) secretion, inducible NO synthase (iNOS) expression and the activation of signal transduction pathways of inflammation, NF-kappaB and the mitogen-activated protein kinases [extracellular signal-regulated kinases (ERK1/2) and C-Jun N-terminal kinase (JNK)], in the A549 lung epithelial cell line. RESULTS: Stimulation with the cytokines interleukin-1beta(IL-1beta)/interferon-gamma (IFN-gamma) increased NO up to 3.3-fold and moxifloxacin inhibited this up to 68% (P < 0.05). Similarly, the increase in iNOS levels was inhibited in cells pre-treated with moxifloxacin by up to 62%. IL-1beta stimulated a rapid increase in the activities of early intracellular signalling molecules, ERK1/2 and JNK. Moxifloxacin inhibited ERK1/2 by up to 100% and p-JNK activation by 100%. NF-kappaB, as measured by electrophoretic mobility shift assay, was inhibited up to 72% by moxifloxacin. Western-blot analysis revealed that IL-1beta enhanced NF-kappaB p65 and p50 proteins by 1.7- and 3.6-fold, respectively, whereas moxifloxacin inhibited the proteins by up to 60%. CONCLUSIONS: Moxifloxacin inhibits intracellular signalling, iNOS expression and NO secretion in a lung epithelial cell line. Future studies may uncover a primary site of quinolone immunomodulation, either upstream or at the cell membrane. Eventually, this quinolone might become an important therapy for inflammatory lung diseases.

Anti-inflammatory effects of moxifloxacin on activated human monocytic cells: inhibition of NF-kappaB and mitogen-activated protein kinase activation and of synthesis of proinflammatory cytokines.

We previously showed that moxifloxacin (MXF) exerts protective anti-inflammatory effects in immunosuppressed mice infected with Candida albicans by inhibiting interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) production in the lung. Immunohistochemistry demonstrated inhibition of nuclear factor (NF)-kappaB translocation in lung epithelium and macrophages in MXF-treated mice. In the present study we investigated the effects of MXF on the production of proinflammatory cytokines (i.e., IL-8, TNF-alpha, and IL-1beta) by activated human peripheral blood monocytes and THP-1 cells and analyzed the effects of the drug on the major signal transduction pathways associated with inflammation: NF-kappaB and the mitogen-activated protein kinases ERK and c-Jun N-terminal kinase (JNK). The levels of IL-8, TNF-alpha, and IL-1beta secretion rose 20- and 6.7-fold in lipopolysaccharide (LPS)-activated monocytes and THP-1 cells, respectively. MXF (5 to 20 microg/ml) significantly inhibited cytokine production by 14 to 80% and 15 to 73% in monocytes and THP-1 cells, respectively. In THP-1 cells, the level of NF-kappaB nuclear translocation increased fourfold following stimulation with LPS-phorbol myristate acetate (PMA), and this was inhibited (38%) by 10 microg of MXF per ml. We then assayed the degradation of inhibitor (I)-kappaB by Western blotting. LPS-PMA induced degradation of I-kappaB by 73%, while addition of MXF (5 microg/ml) inhibited I-kappaB degradation by 49%. Activation of ERK1/2 and the 46-kDa p-JNK protein was enhanced by LPS and LPS-PMA and was significantly inhibited by MXF (54 and 42%, respectively, with MXF at 10 microg/ml). We conclude that MXF suppresses the secretion of proinflammatory cytokines in human monocytes and THP-1 cells and that it exerts its anti-inflammatory effects in THP-1 cells by inhibiting NF-kappaB, ERK, and JNK activation. Its anti-inflammatory properties should be further assessed in clinical settings.