Nucleoplasmic bridges are a sensitive measure of chromosome rearrangement in the cytokinesis-block micronucleus assay

We have performed experiments using the WIL2-NS human B lymphoblastoid cell line and primary human lymphocytes to: (i). determine the importance of including measurements of nucleoplasmic bridges (NPB) in the cytokinesis-block micronucleus (CBMN) assay; (ii). provide evidence that NPB originate from dicentric chromosomes and centric ring chromosomes. In addition, we describe theoretical models that explain how dicentric chromosomes and centric ring chromosomes may result in the formation of NPB at anaphase. The results with WIL2-NS showed that it was possible to distinguish genotoxic effects induced by different oxidizing agents in terms of the NPB/micronucleus frequency ratio. The results with lymphocytes indicated a strong correlation: (i). between NPB, centric ring chromosomes and dicentric chromosomes in metaphases (r > 0.93, P < 0.0001); (ii). between micronuclei (MNi), acentric chromosome fragments and acentric ring chromosomes (r > 0.93, P < 0.0001). The dose-response curves with gamma-rays were very similar for NPB, ring chromosomes and dicentric chromosomes, as were the dose-response curves for MNi, acentric rings and fragments. However, not all acentric chromosomes and dicentric chromosomes/centric rings were converted to MNi and NPB respectively, depending on the dose of radiation. Preliminary data, using FISH, suggest that NPB often represent DNA from a structural rearrangement involving only one or two homologous chromosomes. The results from this study validate the inclusion of NPB in the CBMN assay which provides a valuable measure of chromosome breakage/rearrangement that was otherwise not available in the micronucleus assay. The CBMN assay allows NPB measurement to be achieved reliably because inhibition of cytokinesis prevents the loss of NPB that would otherwise occur if cells were allowed to divide.     

A comparison of folic acid deficiency-induced genomic instability in lymphocytes of breast cancer patients and normal non-cancer controls from a Chinese population in Yunnan

We hypothesized that the genomic response to folate deficiency might be different between breast cancer cases and healthy subjects. To test this hypothesis, we performed a comprehensive study on the genotoxic and cytotoxic effects of in vitro folic acid (FA) deficiency on primary human lymphocytes from 19 breast cancer patients and 20 age-matched healthy females from Yunnan, China using the cytokinesis-block micronucleus assay. Lymphocytes from the volunteers were cultured in RPMI1640 medium containing 30, 120 or 240 nM FA for 9 days. The results showed that 30 nM FA was associated with increased frequencies of micronucleated binucleated cell (MNed BNC), nucleoplasmic bridges (NPB), nuclear buds (BUD), apoptosis (APO) and necrosis (NEC) relative to 120 and 240 nM FA (P<0.001) in lymphocytes of case and control groups in vitro, however there were no significant differences between the 120 and 240 nM FA within each sampling group. The case group showed significantly higher frequencies of MNed BNC than control at 120 and 240 nM FA (P<0.05-0.001) but not at 30 nM FA (P=0.052). NEC was significantly higher in breast cancer group than control at all concentrations of FA (P<0.005). FA concentration explained 60, 39, 39, 52 and 71% of the variance of MNed BNC, NPB, BUD, APO and NEC, respectively compared with breast cancer status which only explained 6 and 7% of the variance of MNed BNC and NEC(Two way ANOVA, P<0.0001). Difference of difference analysis showed that breast cancer cases were not abnormally sensitive to the genome-damaging effect of folate deficiency. We concluded that (i) increased concentrations of FA abolished the genome-damaging effect of FA deficiency in lymphocytes of both breast cancer patients and controls to a similar extent and (ii) FA concentration is much more important than breast cancer status in determining genomic instability and cell death.     

The effect of histamine on the oxidative burst of HL60 cells before and after exposure to reactive oxygen species

During an inflammation neutrophils are stimulated to produce reactive oxygen species (ROS). These ROS induce the release of histamine from mast cells, which are also present at the inflammation site. In this study dibutyryl cAMP differentiated HL60 cells are used as a model for human neutrophils. The effect of histamine on formyl-methionyl-leucyl-phenylalanine (fmlp) stimulated cells is examined. Except for histamine also an accumulation of ROS takes place at the inflammation site and we investigated if ROS can influence the response of the stimulated HL60 cells. It is found that 10^–3 M histamine can inhibit the fmlp induced superoxide anion radical production. This occurs partly via an H₂ receptor because H₂ antagonists like famotidine, mifentidine and ranitidine could partially antagonize this effect of histamine. When HL60 cells are exposed to hydrogen peroxide or hypochlorous acid (20 min), an increased fmlp response is found while the inhibiting effect of histamine remains unchanged.     

Evaluation of the genotoxic effects of the boron neutron capture reaction in human melanoma cells using the cytokinesis block micronucleus assay

The present work reports on the genotoxicity of the boron neutron capture (BNC) reaction in human metastatic melanoma cells (A2058) assessed by the cytokinesis block micronucleus assay (CBMN) using p-borono-L-phenylalanine (BPA) as the boron delivery agent. Different concentrations of BPA (0.48, 1.2 and 2.4 mM) and different fluences of thermal neutrons were studied. Substantial genotoxic potential of alpha and lithium particles generated inside or near the malignant cell by the BNC reaction was observed in a dose-response manner as measured by the frequency of micronucleated binucleated melanoma cells and by the number of micronuclei (MN) per binucleated cell. The distribution of the number of MN per micronucleated binucleated cell was also studied. The BNC reaction clearly modifies this distribution, increasing the frequency of micronucleated cells with 2 and, especially, > or =3 MN and conversely decreasing the frequency of micronucleated cells with 1 MN. A decrease in cell proliferation was also observed which correlated with MN formation. A discrete genotoxic and anti-proliferative contribution from both thermal neutron irradiation and BPA was observed and should be considered secondary. Additionally, V79 Chinese hamster cells (chromosomal aberrations assay) and human lymphocytes (CBMN assay) incubated with different concentrations of BPA alone did not show any evidence of genotoxicity. The presented results reinforce the usefulness of the CBMN assay as an alternative method for assessment of the deleterious effects induced by high LET radiation produced by the BNC reaction in human melanoma cells.     

Xanthine oxidase activity in rat pulmonary artery endothelial cells and its alteration by activated neutrophils.

The possibility that endothelial cell-derived oxidants could contribute to neutrophil-mediated endothelial cell injury and cytotoxicity has been a subject of speculation. Rat pulmonary artery endothelial cells (RPAECs) were examined for the presence of xanthine oxidase (XO) activity, a well-known source of O2-. Using a sensitive assay based on measurements of radioactive xanthine conversion to uric acid by high performance liquid chromatography (HPLC), RPAEC extracts were found to contain both XO and xanthine dehydrogenase (XD) activities. Extracts from early passage cells have 55.3 +/- 11.7 (mean +/- SE) units/10(6) cells of total (XO + XD) activity, one unit of activity being defined as the conversion of 1% of substrate to product in 30 minutes of incubation. XO comprised 31.6 +/- 3.1% of this total activity. Addition of human neutrophils stimulated with phorbol myristate acetate (PMA) caused a rapid and dose-dependent increase in RPAEC XO activity from 31.6 +/- 3.1% to 71.7 +/- 4.8% of total without altering total (XO + XD) activity. The neutrophil dose-response curve for increase in XO paralleled closely the curve for neutrophil-mediated RPAEC cytotoxicity. The basal XO and XD activities and the neutrophil-induced increase in XO activity were inhibited by treating RPAECs with allopurinol, oxypurinol, and lodoxamide, which also inhibited cytotoxicity, but not by catalase, superoxide dismutase, or deferoxamine. Addition of H2O2 failed to cause an increase in RPAEC XO activity or XD to XO conversion. The results suggest that during neutrophil-mediated injury, rapid conversion of RPAEC XD to XO occurs, resulting in increased XO, catalyzed endogenous oxidant production, which may contribute to the oxidant burden in the killing mechanism initiated by activated neutrophils. Although the mechanism for conversion of XD to XO is uncertain, it appears that neutrophil-derived H2O2 is not sufficient to cause this phenomenon. Furthermore, neither O2- nor chelatable iron is required for neutrophil-induced XD to XO conversion. Supernatant fluids from activated neutrophils failed to induce XD to XO conversion in RPAECs. This in vitro system provides an opportunity to define the cellular and molecular mechanisms underlying the in vivo phenomenon of XD to XO conversion associated with ischemic/reperfusion or inflammatory tissue injury.     

Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils

Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H2O2 production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl-methionyl-leucyl-phenylalanine (fMLP) generated mainly extracellular H2O2 and did not undergo apoptosis. Exogenously added H2O2, together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin-induced intracellular H2O2 production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide-differentiated HL-60 cells, which are incapable of intracellular H2O2 production, whereas H2O2 induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H2O2 to commit suicide.     

Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.     

活性氧促进中性粒细胞与骨髓基质细胞黏附及机制初探

目的:探讨活性氧对中性粒细胞与骨髓基质细胞(BMSCs)黏附的影响及其机制。方法:采用密度梯度离心法从小鼠胫骨和股骨分离骨髓中性粒细胞,并用DMSO诱导HL60细胞分化为成熟中性粒细胞(d HL60细胞)。利用分光光度法检测CFDA-SE标记的小鼠骨髓中性粒细胞和d HL60细胞在H_2O_2刺激下与BMSCs的黏附。利用荧光显微成像技术和Western blot检测携带去谷胱甘肽化酶谷氧还蛋白1(Grx1)表达载体的慢病毒感染的d HL60细胞中Grx1的表达。PCR检测Grx1敲除小鼠的基因型。结果:(1) Diff-Quick染色显示,分离的骨髓中性粒细胞纯度高于90%; H_2O_2处理后,中性粒细胞与BMSCs黏附率显著增加(P 0. 01)。(2)荧光显微镜观察和Western blot结果表明,Grx1稳转株d HL60细胞中Grx1的表达水平较空载体稳转株细胞显著增加;体外黏附实验表明过表达Grx1的d HL60细胞较对照组d HL60细胞,在相同H_2O_2浓度下,黏附到BMSCs程度显著下降(P 0. 01)。(3)经PCR鉴定,实验所用基因敲除小鼠在全基因水平上未见Grx1;且敲除Grx1的小鼠较野生型小鼠,在相同浓度H_2O_2刺激下,其骨髓中性粒细胞与BMSCs黏附率显著升高。(4)血管细胞黏附分子1(VCAM-1)抗体预处理BMSCs后,由H_2O_2引起的d HL60与BMSCs之间的黏附增强回复到了静息水平。结论:活性氧促进骨髓中中性粒细胞与BMSCs的黏附,其可能的机制是通过诱导VCAM-1黏附信号的S-谷胱甘肽化。     

Apoptosis-dependent and -independent mechanisms mediate the phagocytic recognition/clearance of the HL60-A1 transfectants with prolonged survival

Objective: The phagocytic recognition and clearance of the recruited inflammatory cells with prolonged survival play a pivotal role in relieving tissue inflammation and maintaining tissue homeostasis. Transgenic mice expressing Bcl-2 in mature neutrophils demonstrated that Bcl-2 attenuated neutrophil apoptosis, while the homeostasis of the neutrophil population was essentially unaffected. This result suggests that clearance of neutrophils with prolonged survival operates independently from apoptosis. Owing to the constitutive and inducible expression of Bcl-2 homologue, A1 in human neutrophils and the intolerance of preparation for the isolated human neutrophils with prolonged survival, the human promyelocytic HL60-A1 transfectants were established to study the mechanism of phagocytic recognition/clearance of the cells with prolonged survival. Materials and Methods: The non-apoptotic cells with prolonged survival were enriched by serum withdrawal for five days and negatively isolated by annexin V-binding beads. Then, the cells were labeled with a fluorogenic marker. Monocyte-derived macrophages (MDM) were co-cultured to perform the phagocytosis assay, and flow cytometry was employed to determine the phagocytic index. Results: In the serum-free condition, the phagocytic index of HL60-A1 transfectants was little different from that of the HL60-EGFP control, despite showing a significantly lower degree of apoptosis. While the phagocytic index of HL60-EGFP control was significantly correlated with the degree of apoptosis, the index of the HL60-A1 transfectants was less relevant to it. The phagocytic index for the annexin V-positive cells did not distinguish the two cell types. However, the phagocytic index for the annexin V-negative cells from the HL60-A1 transfectants was increased with age in days. Preincubation of MDM with the scavenger receptor inhibitor, Oxi-LDL, and the inhibitory antibodies against αvβ3, CD14 and CD36 surface molecules could attenuate the phagocytic recognition of the annexin V-positive HL60 cells but not the annexin V-negative A1 transfectants with prolonged survival. Conclusions: This study thus suggests that a mechanism unrelated to apoptosis exists, which mediates the phagocytic clearance of the non-apoptotic cells with prolonged survival and may be associated with A1 function in the myeloid cells. Received 2 August 2006; returned for revision 7 September 2006; accepted by G. Wallace 29 November 2006 C.-L. Chou and L.-L. Chiang contributed equally to the work on this project as first authors.     

Anti-inflammatory effects of a titanium-peroxy gel: role of oxygen metabolites and apoptosis

Polymorphonuclear neutrophils (PMN) are among the first inflammatory cells to arrive at an implant interface, where they encounter with the foreign material and may produce reactive oxygen species (ROS). During the interaction between titanium and ROS, titanium-peroxy (Ti-peroxy) compounds may be formed. We used a Ti-peroxy gel, made from titanium and hydrogen peroxide, to study the effects of Ti-peroxy compounds on PMN. In the absence of serum, the Ti-peroxy gel decreased the oxidative response of PMN to yeast and PMA and reduced PMN apoptosis without inducing necrosis. These effects could not be ascribed to the release of hydrogen peroxide from the Ti-peroxy gel, because a steady-state hydrogen peroxide producing system failed to mimic the effects of the gel. The effects were similarly unaffected when PMN were preincubated with beta(2)-integrin antibodies, questioning the involvement of adhesion molecules. Nevertheless, when a filter was used to separate the Ti-peroxy gel from the cells, the gel effect on PMN life span was abolished, pointing to a contact-dependent mechanism. In the presence of serum, the Ti-peroxy gel had no effect on the PMN oxidative response and life span, but appeared rather inert. In summary, this study demonstrates that the Ti-peroxy gel has potentially anti-inflammatory properties through a combined peroxide and physical contact effect, supporting the notion that interactions between titanium and inflammatory cells are responsible for the good performance of titanium in vivo.     

Cytotoxicity and genotoxicity of ultrafine crystalline SiO2 particulate in cultured human lymphoblastoid cells

Respirable crystalline silica has been classified as a human lung carcinogen. Ultrafine (diameter < 100 nm) silica particles may be important in carcinogenesis, although the mechanisms remain unclear. In the present study, WIL2-NS cells were incubated for 6, 24, and 48 hr with 0, 30, 60, and 120 microg/ml ultrafine crystalline SiO(2) (UF-SiO(2)). The cytotoxic and genotoxic effects caused by UF-SiO(2) in cultured human cells were investigated via a set of bioassays. Significant dose- dependent decreases in percent cell viability were seen with increasing dose of UF-SiO(2) in the methyl tetrazolium assay. Significant decreases were seen at 120 microg/ml (58, 38, and 57% for 6, 24, and 48-hr exposure, respectively). During 4 days growth in the flasks, there was a slight recovery observed after washing off UF-SiO(2) as measured by the population growth assay. Significant dose-dependent reduction in the cytokinesis block proliferation index was observed by the cytokinesis block micronucleus assay. Treatment with 120 microg/ml UF-SiO(2) for 24 hr produced a fourfold increase in the frequency of micronucleated binucleated cells (MNBNC). The increase in MNBNC was dose-dependent. The lowest dose that gave a statistically significant increase in MNBNC was 30 microg/ml (24-hr treatment), which had cytotoxicity of less than 10%. There was no significant difference in DNA strand breakage as measured by the Comet assay. A significant increase in induced mutant frequency was found at 120 microg/ml as detected by the hypoxanthine guanine phosphoribosyltransferase mutation assay. The results indicate that UF-SiO(2) is cytotoxic and genotoxic in cultured human cells.     

Response of fibroblast cultures from ataxia-telangiectasia patients to reactive oxygen species generated during inflammatory reactions

Cells from patients with ataxia-telangiectasia (AT) are more sensitive than cells from normal individuals to a number of compounds which induce DNA damage via oxygen-derived free radical attack. We tested the hypothesis that AT cells would show a sensitivity to reactive oxygen species (ROS) generated by activated inflammatory cells. AT cells were exposed to neutro-phils activated with 12-O-tetradecanoyl-phor-bol-13-acetate (TPA) or to xanthine/xanthine oxidase (X/XO), an enzyme system which generates superoxide and hydrogen peroxide. Induced micronuclei (MN) frequencies (corrected for spontaneous MN frequencies) were significantly higher in AT cell cultures than in cultures from normal individuals (comparison of MN frequencies of AT vs. normal cultures: for treatment with activated neutrophils, P = 0.003; for X/XO, P = 0.05). The comet assay was used to determine whether the elevated chromosomal damage in the treated AT cells was due to a difference in strand breakage or its rejoining. X/XO treatment was used in studies of single-stranded (SS) DNA breakage, and X-ray treatment for double-stranded (DS) DNA damage. AT and normal cells showed no significant differences in the initial levels of SS (P = 0.29) or DS (P = 0.91) DNA damage. Likewise, they exhibited similar rejoining kinetics (rejoining half-time for SS = 10 min, for DS = 30 min). These data support the involvement of the AT loci in determining a cell's ability to deal with oxidative stress, although the mechanism underlying this effect has yet to be resolved. The data also suggest that AT patients are at elevated risk of sustaining DNA damage in tissues undergoing inflammatory reactions. © 1994 Wiley-Liss, Inc.     

Oxygen radical generation and expression of NADPH oxidase genes in bottlenose dolphin (Tursiops truncatus) neutrophils

Oxygen radical generation by stimulation with phorbol myristate acetate (PMA) was evaluated in bottlenose dolphin neutrophils. A Cypridina luciferin analog-dependent chemiluminescent assay demonstrated that dolphin neutrophils generate superoxide by the addition of PMA, and that its superoxide-forming activity is completely suppressed by diphenylene iodonium, a specific inhibitor of NADPH oxidase. These results indicate that dolphin neutrophils possess NADPH oxidase activity. Furthermore, the NADPH oxidase activity (hydrogen peroxide production) in dolphin neutrophils, as well as in human neutrophils, was greater at 37 degrees C than at a lower temperature. RT-PCR with specific primers revealed that dolphin neutrophils expressed the mRNAs of the major NADPH oxidase components, which included membrane-associated flavocytochrome b (gp91(phox) and p22(phox)) and cytosolic factors (p40(phox), p47(phox), and p67(phox)), implying the existence of these protein homologues in dolphin neutrophils.     

Calcium is required for PMA induced superoxide release from human neutrophils

Experiments were designed to reexamine the relationship between extracellular calcium and superoxide generation in phorbol myristate acetate (PMA) stimulated neutrophils exploiting a newly adapted method to measure superoxide anion (O2-) generation from adherent cells stimulated at high and low cell density. Human neutrophils were plated in microtiter wells in cell densities of either 0.2 or 2.0 million cells/well. Superoxide release was measured sequentially over 60 min by reduction of ferricytochrome c. Cells were maintained in 1 mM Ca++ or 0 mM Ca++ Hanks' buffer for 60 min prior to activation as well as during measurement of O2-. In 1 mM Ca++, 2.0 million adherent neutrophils released 10.7 +/- 1.2 nmol O2- in 20 min (n = 4). O2- release was not significantly different for high density cells incubated and stimulated in 0 mM Ca++. In the presence of 1 mM Ca++, 0.2 million adherent neutrophils released 6.3 +/- 0.5 nmols O2- in 20 min. With cells stimulated at low density, PMA stimulated O2- release was significantly decreased (3.0 +/- 0.6 nmol O2- in 20 min) as was the initial rate of secretion of O2- in the absence of extracellular calcium. Basal release of superoxide was also greater in the presence of 1 mM Ca++ (0.96 nmol/20 min) compared to basal release in 0 mM Ca++ (0.22 nmol/20 min). Additional experiments with 0.2 million cells/well showed that extracellular Ca++ was required during stimulation with PMA and that prior incubation of cells for up to 60 min in 0 mM Ca++ had no effect on O2- release measured in the presence of calcium. Furthermore, PMA stimulated O2- was independent of verapamil (10(-5)-10(-7) M), suggesting that voltage-dependent calcium channels do not participate in this response. The planar areas for unstimulated neutrophils in 0 mM Ca++ increased after addition of PMA. Unstimulated cells in 1 mM Ca++ tended to be larger and planar areas did not increase after PMA. These studies demonstrate that PMA stimulated O2- secretion is dependent on extracellular calcium particularly when adherent neutrophils are stimulated at low cell density. Furthermore, extracellular calcium at a concentration of 1 mM primes neutrophils by increasing basal secretion of O2- and increasing superoxide release after a maximum stimulating dose of PMA.     

Phorbol myristate acetate-induced modulation of antibody-dependent cellular cytotoxicity by human polymorphonuclear leukocytes.

The effect of phorbol myristate acetate (PMA) on the antibody-dependent cellular cytotoxicity (ADCC) of human polymorphonuclear leukocytes (PMNs) against human erythroleukemic K562 cells was studied by the use of a 3-h 51Cr-release assay. Pretreatment of PMNs with PMA (10 ng/ml) for 60 min resulted in inhibition of subsequent ADCC. This inhibition was dependent on doses of PMA. The effect of pretreatment of PMNs with PMA on O2- generation of the cells was also studied. The ability of the cells to generate O2- was not suppressed, and the expression of Fc receptors on the cell membrane was well preserved. In contrast, the addition of PMA to the ongoing ADCC (5 to 30 min after the start of the ADCC assay) enhanced the activity of the cells for ADCC. This augmentation was abolished by catalase, whereas ADCC itself was not affected by the agent. These results imply divalent effects of PMA on the ADCC of PMNs. The suppression of ADCC activity of PMNs by pretreatment with PMA is not due to inhibition of the reactive oxygen burst of the cells. The augmentation of ongoing ADCC by the addition of PMA is due to secretion of hydrogen peroxide from the cells induced by PMA, and this augmentation occurs only when the interaction between effector and target cells exists through Fc receptor.     

Co-cultures of Human Coronary Smooth Muscle Cells and Dimethyl Sulfoxide-differentiated HL60 Cells Upregulate ProMMP9 Activity and Promote Mobility—Modulation By Reactive Oxygen Species

Vascular cells and leukocytes, involved in the development of atherosclerosis, produce cytokines and/or reactive oxygen species (ROS) and matrix metalloproteinases (MMPs) implicated in cell mobility. We investigated by co-culture experiments the effects of human coronary smooth muscle cells (HCSMC) on MMPs characteristics and mobility of neutrophil-like dimethyl sulfoxide-differentiated HL60 cells (≠HL60). The effects of superoxide dismutase (SOD) and catalase were also analyzed. All the studied MMP2 characteristics remained unchanged. HCSMC stimulated MMP9 protein level, activity and mobility of ≠HL60 cells and expressed and secreted a variety of cytokines implicated in atherosclerosis. SOD and catalase increased MMP9 expression, protein level and activity of ≠HL60, but migration of ≠HL60 cells was only decreased by catalase, demonstrating that ROS are more efficient in modulating MMP9 activity of ≠HL60 than their mobility. Finally, HCSMC being able to stimulate ≠HL60, their co-cultures may represent an in vitro approach to study cellular interactions occurring in vivo during atherosclerosis.     

Response of the rabbit VH subgroups a and y following antigenic stimulation.

Phorbol myristate acetate (PMA)-activated neutrophils were found to destroy B lymphoblast tumour cells (Raji) as determined by the 51Cr release assay. The target cell lysis was prevented by azide, suggesting the involvement of the myeloperoxidase enzyme. Catalase and cytochrome c caused a marked impairment of the neutrophil-mediated cytolysis, whereas superoxide dismutase significantly enhanced the target cell destruction. These data indicate that hydrogen peroxide plays a key role in the target cell injury; superoxide anion appears to be devoid of direct cytotoxic activity, despite its requirement as a precursor of hydrogen peroxide. The target cell destruction required the presence of the iodide ion as oxidizable co-factor for the myeloperoxidase-hydrogen peroxide system. The chloride ion alone was uneffective. Inhibition of target cell metabolic pathways, involved in the cellular defences against oxidative injury, by the anti-neoplastic agent 1,3-bis-(2-chloroethyl)-1-nitrosurea (BCNU) resulted in an increased neutrophil-mediated cytolysis. Under the experimental conditions employed, PMA-activated neutrophils incubated with BCNU-treated Raji cells became cytotoxic also in the presence of the chloride ion alone as myeloperoxidase co-factor. Our results suggest that Raji target cell destruction by PMA-activated neutrophils depends on the myeloperoxidase-hydrogen peroxide-halide system. The cytolytic event is influenced by target cells themselves, which should be regarded as an active component of the cytotoxic system, capable of interfering with the lytic mediators of the effector cells.     

Necrosis, apoptosis, cytostasis and DNA damage in human lymphocytes measured simultaneously within the cytokinesis-block micronucleus assay: description of the method and results for hydrogen peroxide

A method is described for the inclusion of apoptotic and necrotic cells in the cell counts obtained in the cytokinesis-block micronucleus (CBMN) assay, which is conventionally used solely for the assessment of chromosome breakage, chromosome loss and frequency of dividing cells. The morphological criteria for the recognition and discrimination between necrotic, apoptotic and viable cells are described. Using this comprehensive method we have evaluated the cytotoxic and genotoxic effects of hydrogen peroxide (0-100 microM) in lymphocytes exposed in RPMI 1640 medium. The results obtained indicated significant (P < 0.05) correlations between hydrogen peroxide concentration and the frequency of micronucleated cells (r = 0.39), necrotic cells (r = 0.73), apoptotic cells (r = -0.26) and binucleated cells (r = -0.55). Almost similar results were obtained using the cytosine arabinoside modification of the CBMN assay, which enables excision-repaired sites to be converted to micronuclei. Some of the above end-points were significantly (P < 0.05) correlated with each other (necrosis and apoptosis, R = -0.39; necrosis and micronucleated cell frequency, R = 0.46; necrosis and binucleated cells, R = -0.78; apoptosis and binucleated cells, R = 0.32). It was therefore necessary to use multiple regression analysis to identify the main event induced by hydrogen peroxide, which was necrosis (beta = 0.57, P = 0.0001) and not micronucleus formation (beta = 0. 15, P = 0.1332). Using an ELISA assay we showed that hydrogen peroxide did not induce 8-hydroxydeoxyguanosine. Our data show that the proposed comprehensive test system may provide a better procedure for classifying potential toxic chemicals and enable discrimination between agents that primarily induce cytotoxic effects as opposed to genotoxic effects. The integration of apoptosis and necrosis into the micronucleus assay may also be of practical use in radiosensitivity studies.     

Tumour necrosis factor (TNF)-alpha primes murine neutrophils when triggered via formyl peptide receptor-related sequence 2, the murine orthologue of human formyl peptide receptor-like 1, through a process involving the type I TNF receptor and subcellular granule mobilization

Neutrophil granulocytes play an important role in innate host defence against microbial invasions and they are also the key effector cells in mediating host tissue damage. These functions often rely on the production of reactive oxygen species (ROS) from the membrane-bound NADPH-oxidase system. The magnitude of ROS production varies depending on the state of the cells, i.e. resting or primed. Many priming agents as well as potent NADPH-oxidase activators have been identified and characterized for human neutrophils. The cytokine tumour necrosis factor (TNF)-alpha is one prominent example of a priming agent and the synthetic hexapeptide WKYMVm is an agonist that triggers an activation of the NADPH-oxidase of human neutrophils through two members of the formyl peptide family of receptors, formyl peptide receptor (FPR) and FPR-like 1 (FPRL1). This peptide also activates murine neutrophils but the precise receptor involved has not been previously characterized. We show in this study that WKYMVm activates stably transfected HL60 cells expressing murine formyl peptide receptor-related sequence 2 (Fpr-rs2) and that activation of murine neutrophils with WKYMVm is blocked by an FPRL1-specific antagonist. WKYMVm is thus an agonist for Fpr-rs2 and we suggest that this receptor is in fact the mouse orthologue of FPRL1. In addition, we show that the WKYMVm response in murine neutrophils can be primed by TNF-alpha and this priming process involves mobilization of subcellular granules. The results obtained using neutrophils derived from TNF receptor type I (TNFRI)-deficient animals suggest that TNF-alpha exerts its priming effect via the TNFRI.     

Development of a respiratory burst assay using zebrafish kidneys and embryos

The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including the respiratory burst of phagocytes. Respiratory burst can be used as a reliable measure of the immune response of a host, and numerous assays have been developed to measure this response in a variety of mammal and fish species. Phagocytes, like granulocytes and macrophages, that are derived from different tissues, or grown in cell culture, have been employed in a range of assay formats employing a variety of detection methods. The small size of the zebrafish has prevented the large-scale extraction of these cells for respiratory burst assays in the zebrafish. In this work, we describe a respiratory burst assay developed for the zebrafish using intact kidneys and embryos as sources of phagocytes. Phorbol myristate acetate (PMA)-inducible reactive oxygen species (ROS) were detected following the oxidation of a non-fluorescent dye 2',7'-dihydrodichlorofluorescein diacetate (H2DCFDA) to dichlorofluorescein (DCF), a fluorescent product. Embryos from 1 day post-fertilization until 5 days post-fertilization (dpf) were employed in this assay. Abrogation of H2DCFDA oxidation by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BisI) indicated a reduction in the respiratory burst. Fluorescence from the PMA-induced respiratory burst in kidneys and embryos was significantly elevated above DMSO-treated controls, while preincubation with BisI inhibited the increase in fluorescence. Colocalization of cell-associated chloromethyl-dihydrodichlorofluorescein diacetate (CM-H2DCFDA) with the phagocyte-selective dye neutral red is consistent with the observation that macrophages and granulocytes are the ROS-producing cells in the zebrafish.