Drora Halperin

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

ABSTRACT 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-α) production in the lung. Immunohistochemistry demonstrated inhibition of nuclear factor (NF)-κB 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-α, and IL-1β) 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-κB and the mitogen-activated protein kinases ERK and c-Jun N-terminal kinase (JNK). The levels of IL-8, TNF-α, and IL-1β secretion rose 20- and 6.7-fold in lipopolysaccharide (LPS)-activated monocytes and THP-1 cells, respectively. MXF (5 to 20 μg/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-κB nuclear translocation increased fourfold following stimulation with LPS-phorbol myristate acetate (PMA), and this was inhibited (38%) by 10 μg of MXF per ml. We then assayed the degradation of inhibitor (I)-κB by Western blotting. LPS-PMA induced degradation of I-κB by 73%, while addition of MXF (5 μg/ml) inhibited I-κB 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 μg/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-κB, ERK, and JNK activation. Its anti-inflammatory properties should be further assessed in clinical settings.

Ascorbic Acid Inhibits Apoptosis Induced by X Irradiation in HL60 Myeloid Leukemia Cells

Exposure of cells to ionizing radiation can cause apoptosis. Since antioxidants have been shown to protect against radiation-induced apoptosis, in this study we have evaluated the putative protective effect of ascorbate against radiation-induced apoptosis as well as the production of peroxides in the cells. HL60 cells transport the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulate reduced ascorbate. Exposure of the cells to 5-40 Gy X radiation resulted in induction of apoptosis. Preincubation of the cells with DHA reduced the level of apoptosis after exposure to 5-20 Gy. Exposure of the cells to 5 or 20 Gy X radiation did not affect the intracellular concentration of peroxides, while phorbol myristate acetate (PMA), which is known to induce production of H(2)O(2) in cells (and served as a control), resulted in an increase in peroxides and a decrease in intracellular ascorbate. Irradiation of the cells with 1-3 Gy resulted in up-regulation of expression of BCL2 without affecting the level of apoptosis. At higher doses of radiation, enhanced BCL2 expression did not prevent radiation-induced apoptosis. Loading of the cells with ascorbate prior to their exposure to 1-3 Gy X radiation did not affect the enhanced BCL2 expression observed in the irradiated cells. At higher doses of radiation, ascorbate decreased apoptosis and restored the level of BCL2 in the cells. Exposure of the cells to 3-20 Gy X radiation enhanced the cell surface expression of TNFRSF6 (formerly known as Fas/APO-1) antigen and enhanced anti-TNFRSF6 antibody-induced apoptosis of the cells. Ascorbate loading did not affect expression of TNFRSF6 and did not overcome the anti-TNFRSF6 antibody-induced apoptosis. In conclusion, our data demonstrate that exposure of HL60 cells to radiation enhanced BCL2 and TNFRSF6 expression. Ascorbate did not affect BCL2 or TNFRSF6 expression. We therefore conclude that it protects HL60 cells against radiation-induced apoptosis, although the mechanisms of protection must still be elucidated.

Immunomodulatory effects of moxifloxacin in comparison to ciprofloxacin and G‐CSF in a murine model of cyclophosphamide‐induced leukopenia

Abstract: We analyzed the effect of the two quinolones moxifloxacin and ciprofloxacin on the repopulation of hematopoietic organs and on the production of cytokines by various organs of cyclophosphamide (CP)‐induced leukopenic mice. The effect was compared to that of G‐CSF. Cyclophosphamide injection induced a severe leukopenia, with nadir at day 4 post‐injection. All the quinolone and G‐CSF‐treated animals showed WBC>500/μL at the nadir, compared to 50% of saline‐treated mice. Cyclophosphamide induced a marked decrease in the number of myeloid progenitors (CFU‐C) in bone marrow (BM) and spleen. Quinolone or G‐CSF treatment resulted in a 1.4–4.3‐fold increase in CFU‐C numbers in the BM; no enhancement was observed in the spleen. Treatment with CP resulted in enhanced colony‐stimulating activity (CSA) in bone shaft and spleen and decreased activity in bladder and lung. Treatment of CP‐injected mice with quinolones significantly enhanced CSA in the bone shaft, spleen, lung and bladder on different days.

Immunomodulatory effects of ciprofloxacin in TNBS-induced colitis in mice

Background Crohns exacerbation and pouchitis are commonly treated with ciprofloxacin and metronidazole. Few studies have shown an advantage of this regimen compared with other antibiotics. Most attributed the effect to its better antibacterial coverage. Others have shown an apparent anti‐inflammatory effect of quinolones in several in vitro and in vivo models of inflammation other than inflammatory bowel diseases (IBD). Our objective was to test the hypothesis that ciprofloxacin may act as an anti‐inflammatory agent rather than just an antibacterial drug using a model of chemical colitis. Methods TNBS colitis was induced in BALB/c mice. The anti‐inflammatory effect of ciprofloxacin compared with ceftazidime and dexamethasone was assessed. Results Mice treated with ciprofloxacin (7.5 mg/kg or 15 mg/kg) had significant reductions in clinical signs, body weight loss, splenic and colonic weight increase compared with saline‐treated and ceftazidime‐treated mice. Histologic analysis showed mild inflammation in ciprofloxacin‐treated mice with a mean score of 3.8 ± 0.5 points compared with moderate colitis scored 7.8 ± 1.3 and 9.5 ± 0.5 points in saline and ceftazidime‐treated mice, respectively. Analysis of cytokine levels in colon homogenates showed a significant decrease of IL‐1&bgr;, IL‐8, and TNF&agr; levels in ciprofloxacin‐treated animals. Immunohistochemistry for NF&kgr;B showed strong positivity in saline and ceftazidime‐treated mice in contrast to weak focal stain in ciprofloxacin‐ and dexamethasone‐treated mice. Conclusions These findings imply that ciprofloxacin has an anti‐inflammatory effect, rather than just an antibacterial one, making its use favorable in IBD patients. (Inflamm Bowel Dis 2007)

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)-&ggr; 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-&ggr; and LPS induced higher levels of TLR4 expression than stimulation with IFN-&ggr; alone. Using the protein synthesis inhibitor cycloheximide and measuring the expression of CD35 in neutrophils stimulated with IFN-&ggr; and LPS alone or in combination, we could demonstrate that IFN-&ggr; 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-&ggr; and LPS induced neutrophil activation and enhanced secretion of the cytokines, interleukin (IL)-8, IL-1&bgr;, tumor necrosis factor-&agr;, 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-&ggr; primes neutrophils to respond to LPS.

Oxidative stress causes telomere damage in Fanconi anaemia cells - a possible predisposition for malignant transformation.

Fanconi anaemia (FA) is an autosomal recessive and X‐linked disease characterized by severe genetic instability and increased incidence of cancer. One explanation for this instability may be the cellular hypersensitivity to oxidative stress leading to chromosomal breaks. This study explored the possible oxidative damage to telomeres of FA lymphocyte cell line, HSC536/N, and its possible effect on telomere function. We postulated that combination of oxidative damage with overexpression of telomerase may provide a possible model for malignant transformation in FA. The cells were grown in the presence of telomerase inhibitor and exposed for 1 month to H2O2 combined with various antioxidants. This exposure caused shortening of telomere length and damage to the telomere single stranded overhang, which was prevented by several oxidants. This shortening was associated with development of severe telomere dysfunction. Control cells did not exhibit this sensitivity to H2O2. Telomere dysfunction did not evoke damage response in FA cells, in contrast to normal P53 upregulation in control cells. Reconstitution of telomerase activity protected FA telomeres from further oxidative damage. These results suggest a scenario in which oxidative stress causes telomere shortening and ensuing telomere dysfunction may form the basis for malignant transformation in FA cells. Upregulation of telomerase activity in sporadic FA cells may perpetuate that process, thus explaining the malignant character of FA cells in vivo.

Moxifloxacin enhances antiproliferative and apoptotic effects of etoposide but inhibits its proinflammatory effects in THP-1 and Jurkat cells.

Etoposide (VP-16) is a topoisomerase II (topo II) inhibitor chemotherapeutic agent. Studies indicate that VP-16 enhances proinflammatory cytokines secretion from tumour cells, including IL-8, a chemokine associated with proangiogenic effects. Fluoroquinolones inhibit topo II activity in eukaryotic cells by a mechanism different from that of VP-16. The fluoroquinolone moxifloxacin (MXF) has pronounced anti-inflammatory effects in vitro and in vivo. We studied the effects of MXF and VP-16 on purified human topo II activity and further analysed their combined activity on proliferation, apoptosis and caspase-3 activity in THP-1 and Jurkat cells. Moxifloxacin alone slightly inhibited the activity of human topo II; however, in combination with VP-16 it led to a 73% reduction in enzyme activity. VP-16 inhibited cell proliferation in a time and dose-dependent manner. The addition of moxifloxacin for 72 h to low-dose VP-16 doubled its cytotoxic effect in THP-1 and Jurkat cells (1.8- and 2.6-fold decrease in cell proliferation, respectively) (P<0.004). Moxifloxacin given alone did not induce apoptosis but enhanced VP-16-induced apoptosis in THP-1 and Jurkat cells (1.8- and two-fold increase in annexin V positive cells and caspase-3 activity, respectively) (P<0.04). VP-16 induced the release of IL-8 in a time and dose-dependent manner from THP-1 cells. Moxifloxacin completely blocked the enhanced release of IL-8 induced by 0.5 and 1 μg ml−1 VP-16, and decreased IL-8 release from cells incubated for 72 h with 3 μg ml−1 VP-16 (P<0.001). VP-16 enhanced the release of IL-1β and TNF-α from THP-1 cells, whereas the addition of MXF prevented the enhanced cytokine secretion (P<0.001). We conclude that MXF significantly enhances VP-16 cytotoxicity in tumour-derived cells while preventing VP-16-induced proinflammatory cytokine release. This unique combination may have clinical benefits and cytotoxic drug ‘sparing effect’ and should be further studied in vivo.

Moxifloxacin increases anti-tumor and anti-angiogenic activity of irinotecan in human xenograft tumors

Camptothecins (CPTs) are topoisomerase I inhibitors chemotherapeutic agents used in combination chemotherapy. We showed previously that combination of moxifloxacin (MXF) and CPT induced inhibitory effects on topoisomerase I activity, on proliferation of HT-29 cells in vitro and enhanced apoptosis, compared to CPT alone. Analysis of secretion of the pro-angiogenic factors IL-8 and VEGF showed significant reduction by MXF. Using a murine model of human colon carcinoma xenograft, we compared the effects of MXF/CPT in vitro to MXF/irinotecan combination in vivo. We show that the MXF/CPT inhibitory effects observed in vitro are reflected in the inhibition of the progressive growth of HT-29 cells implanted in SCID mice. Using caliper measurements, Doppler ultrasonography, image analyses and immunohistochemistry of nuclear proteins (Ki-67) and vascular endothelial cells (CD-31) we show that addition of MXF (45mg/kg) to a relatively ineffective dose of irinotecan (20mg/kg), results in a 50% and 30% decrease, respectively, in tumor size and a decrease in Ki-67 staining. Power Doppler Ultrasound showed a significant, pronounced decrease in the number of blood vessels, as did CD-31 staining, indicating decreased blood flow in tumors in mice treated with MXF alone or MXF/irinotecan compared to irinotecan. These results suggest that the combination of MXF/irinotecan may result in enhanced anti-neoplastic/anti-angiogenic activity.

Quinolones as enhancers of camptothecin-induced cytotoxic and anti-topoisomerase I effects

Camptothecins (CPTs) are topoisomerase I (topo I) inhibitor chemotherapeutic agents. Studies indicate that combination therapy is needed in most therapeutic protocols with camptothecins. Certain fluoroquionolones inhibit topoisomerase II activity in eukaryotic cells. We showed previously that the fluoroquionolone moxifloxacin inhibited purified human topoisomerase II, acted synergistically with etoposide and enhanced anti-proliferative effect in THP-1 and Jurkat cells. There is no information on flouroquionolones activity on topoisomerase I. We examined the effect of moxifloxacin and ciprofloxacin alone or in combination with camptothecin on purified topoisomerase I activity and further analysed their combined activity on proliferation and apoptosis in HT-29 cells. Moxifloxacin and ciprofloxacin alone slightly inhibited purified topoisomerase I activity; however in combination with camptothecin it led to a 82% and 64% reduction in enzyme activity, respectively. Moreovwer, our studies indicate that incubation of HT-29 cells with a combination of moxifloxacin or ciprofloxacin with CPT increases cellular topoisomerase I inhibitory activity. In cell proliferation assays, addition of moxifloxacin to 1nM camptothecin enhanced its cytotoxic activity by three-fold and was similar to that of 50nM camptothecin alone (45+/-2.1% inhibition). Ciprofloxacin enhanced cytotoxic activity to a lesser extent. Apoptosis studies showed up to 1.6-fold increase in annexin V positive cells when the fluoroquinolones were combined with camptothecin as compared to camptothecin alone. Analysis of the proangiogenic factors IL-8 and VEGF showed significant reduction in IL-8 production by moxifloxacin and ciprofloxacin up to 48% and in VEGF secretion from the cells. Further in vivo and clinical studies of camptothecins combined with the above fluoroquinolones are warranted.

Effects of the methanol extract residue (MER) tubercle bacillus fraction on the production of antibodies in vitro: II. Effects on macrophage and lymphocyte populations

The effect of the methanol extract residue (MER) fraction of BCG tubercle bacilli on the generation of primary antibody responsiveness in vitro to sheep red blood cells (SRBC) was ascertained in cell reconstitution experiments, employing enriched populations of mouse macrophages and of T and B lymphocytes. In each of the antibody generation cultures one or another of the cell fractions had been exposed to MER, either by treatment of the donor animals or by preincubation with the agent for 48 hr in vitro. In some experiments, supernatants of MER-preincubated cells were employed in place of the cells. Macrophages and T cells that had been exposed to MER in vivo or in vitro and their supernatants demonstrated a markedly greater effect than nonexposed cells in the generation of direct specific plaque-forming cells (PFC) upon antigenic stimulation of the cultures with SRBC. In contrast, PFC production was not stimulated in B-lymphocyte populations that had been in contact with the agent.