Release Of Oxygen Intermediates Research Articles

Responses of tropical tree species to urban air pollutants: ROS/RNS formation and scavenging

Air pollution in an urban environment is the major stress factor for vegetation due to the direct generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). To quantify urban air pollution-induced ROS/RNS formation, damage and detoxification, nine different biochemical parameters related to free radical formation, scavenging and membrane damage were estimated in twelve tropical tree species. The experiment was performed in three different seasons at four distinct urban environments in Varanasi city located in the Indo-Gangetic plain of India. Redundancy analysis was performed to statistically assess the relationship between air pollutants (PM2.5, NO2, SO2 and O3) and temperature with ROS/RNS generation and their detoxification. Significant effects of air pollution exposure and temperature on ROS/RNS formation, scavenging and membrane damage were recorded with increasing pollution load in the city for all the tree species. The extent of variability (47–87%) in responses of different tree species was due to their intrinsic ability to scavenge free radicals which minimized the membrane damage. PM2.5, NO2 and O3 were identified as major pollutants that influenced trees to different extents in regulating ROS/RNS. However, the response was maximum against NO2 (34–72%) followed by PM2.5 (16–64%) and O3 (3–31%), indicating that under urban environment, trees are considerably sensitive to the combined effects of both particulate and gaseous pollutants. Reactive oxygen intermediate release, total free radical scavenging activity, NO scavenging activity and membrane stability index were identified as major parameters which showed distinct responses with increasing pollution load. Caesalpinia sappan, Ficus religiosa and Albizia lebbeck were identified as most tolerant tree species having higher ROS/RNS scavenging potential resulted in lower membrane damage. Thus responses of urban trees to air pollution are governed by their intrinsic defence mechanisms to scavenge ROS/RNS by maintaining the membrane integrity through integrated cross-talk between different antioxidative pathways.

Toll-Like Receptor 2 Signalling Regulates Oxidative Burst of Human Neutrophils, but Not Cytokine Response of Dendritic Cells after Contact with Aspergillus fumigatus.

Toll-like receptors (TLRs) play a crucial role for detecting conserved pathogen-associated molecular patterns comprising fungal structures. Activation of polymorphonuclear granulocytes (PMNs) as well as mobilization of antigen presenting cells is important for successful clearance of fungal infections. Here, the role of TLR2 and TLR4 for activation of PMNs and immature dendritic cells (iDCs) through Aspergillus fumigatus was analyzed.Generation of iDCs was achieved by cultivating monocytes in the presence of GM-CSF and IL-4. PMNs were obtained from fresh blood. In blocking studies, cells were preincubated with anti-TLR2 and / or anti-TLR4 antibody before stimulation with Aspergillus fumigatus germinating conidia. For RNA interference (RNAi) experiments, iDCs were transfected with short-interfering RNA (siRNA) via electroporation and gene expression was controlled by quantitative real-time PCR.Oxidative burst of PMNs was induced after contact with Aspergillus or TLR2 ligand zymosan. Release of oxygen intermediates could be significantly reduced if PMNs were preincubated with anti-TLR2 antibody.In contrast to monocytes, cytokine secretion (IL-12 and TNF-α) of iDCs was not altered if anti-TLR antibodies were used. To study the role of TLR2 and TLR4 more detailed, an RNAi system for iDCs was established to downregulate gene expression. Determination of siRNA transfection rate revealed an efficiency of about 85% and allowed significant downregulation of TLR2 and TLR4 (> 90%). In accordance to blocking studies, expression of IL-10, IL-12 and TNF-a was not reduced after TLR2 or TLR4 siRNA transfection and stimulation with Aspergillus.We conclude that TLR2 plays an important role in the induction of the oxidative burst of PMNs whereas cytokine release of iDCs seems to be independent of TLR2 / TLR4 signalling. The established siRNA system allows the detection of receptors responsible for activation of iDCs.

Interaction between leucocytes and human spermatozoa influencing reactive oxygen intermediates release.

The relationship between the presence of white blood cells (WBCs) and the fertilizing potential of human semen is still an open question. It is well known that the presence of leucocytes in human semen can be related to the production of reactive oxygen intermediates (ROI). Semen samples were obtained from 15 normozoospermic men and leucocytes were isolated from heparinized blood drawn from 15 volunteers. Lucigenin and luminol-mediated chemiluminescence assays were used to determine reactive oxygen species (ROS) generation by non-activated or activated leucocytes through 12-myristate-13-acetate or N-formyl-methionyl-leucyl-phenyalanine (FMLP) before the addition of spermatozoa isolated by swim-up or Percoll procedures. All spermatozoal fractions used in this study were characterized by defining their motility, morphology and viability. The levels of ROS formation by non-activated as well as stimulated leucocytes were significantly decreased after addition of swim-up separated spermatozoa (p < 0.01). The ability to inhibit the basal chemiluminescence was of lower degree for spermatozoa isolated from 90% Percoll fractions than for swim-up sperm. However, addition of sperm cells from 47% Percoll fraction was found to increase both lucigenin and luminol signals. Moreover, the determined ROI levels changed depending on the type of inducing factor used for oxidative burst. Then, spermatozoa selected by swim-up procedure although with only slightly higher viability and morphology than sperm obtained from 90% Percoll fraction clearly exhibited much higher capacity to inhibit ROI secretion by receptor-stimulated leucocytes (FMLP-activation) than Percoll fractionated sperm. Such results may indicate that within normal semen may exist sperm subpopulations with different biochemical mechanisms controlling the interaction between spermatozoa and contaminating leucocytes. When ROI levels contained in normozoospermic semen are dependent on the WBCs activation, it seems that spermatozoa with preserved normal functional competence are able to defend themselves against leucocytes-derived ROI. Also for normozoospermic ejaculates, swim-up sperm may improve semen antioxidant characteristics when comparing with Percoll (90%) separated sperm. It may help for optimal sperm preparation when assisting to infertility treatment.

Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor Fc gamma RI (CD64) expressed on human blood dendritic cells.

The mechanisms responsible for efficient sequestration of Ag by cells of the dendritic cell (DC) lineage remain incompletely characterized. One pathway, internalization of Ag-IgG complexes via CD32 (the type II IgG FcR, Fc gamma RII) enhances Ag presentation 100-fold over noncomplexed Ag. Blood leukocytes differentially express two additional IgG FcR, Fc gamma RI (CD64) and Fc gamma RIII (CD16), which may also participate in leukocyte functions such as phagocytosis, Ab-dependent cellular cytotoxicity (ADCC), release of oxygen intermediates, and enhancement of Ag presentation. A phagocytically active form of CD64 was recently demonstrated on human blood DC, but complete functional potential of CD64 on the DC lineage remains undefined. Therefore, highly purified human blood DC (CD33(2)+, CD14-, CD11c2+, HLA-DR3+, CD64+ (CD83+ after overnight culture)) and monocytes (CD33(2)+, CD14(3)+, CD11c2+, HLA-DR+, CD64(2)+, CD83-) were compared for cytokine modulation and effector functions of CD64. Both DC and monocyte CD64 expression was increased by IFN-gamma and IL-10, but while monocyte CD64 was decreased by IL-4, DC CD64 remained unchanged. FcR-mediated functional differences were also evident between the DC and the monocytes. Monocytes generated robust Fc gamma R-dependent superoxide anion release and ADCC activity, while DC failed to release reactive oxygen intermediates and demonstrated minimal ADCC activity, despite apparently normal expression of the gamma-chain subunit and the signaling molecule Syk. In contrast, DC were more efficient than monocytes with respect to T cell activation when Ag was targeted specifically to CD64. These new findings suggest a previously unappreciated potential for CD64 to shape the immune response by dramatically increasing the efficiency with which DC sequester Ag prior to achieving full T cell stimulatory potential.

Dehydroepiandrosterone modulation of lipopolysaccharide-stimulated monocyte cytotoxicity.

Dehydroepiandrosterone (DHEA), the predominant androgen secreted by the adrenal cortex, can be converted to both potent androgens and estrogens. In addition to its role as a precursor for other steroid hormones, DHEA has been proposed to play an important role in immunity. This study has investigated DHEA modulation of LPS-induced monocyte cytotoxicity. Cytotoxicity markers assessed include tumor cell killing, IL-1 secretion, reactive oxygen intermediate release, nitric oxide synthetase activity as measured by the release of reactive nitrogen intermediates, complement receptor-1 cell surface protein, and TNF-alpha protein presence. Monocytes stimulated with LPS concentrations of 1.0 micrograms/ml displayed the above cytotoxic markers, whereas monocytes stimulated with DHEA alone or with LPS at a lower concentration of 0.2 ng/ml did not. However, when used simultaneously, DHEA and LPS 0.2 ng/ml displayed a synergistic effect on monocyte cytotoxicity against cancerous cell lines, IL-1 secretion, reactive nitrogen intermediate release, complement receptor-1 cell-surface protein, and TNF-alpha protein to levels comparable with levels obtained using LPS 1.0 microgram/ml. Finally, Scatchard plot analysis demonstrated the presence of a DHEA receptor in monocytes, suggesting that DHEA effects on LPS-stimulated monocytes are mediated through a receptor-dependent process.

Time course of reactive oxygen intermediates release and histopathological findings during experimental autoimmune prostatitis development.

Spontaneous and stimulated reactive oxygen intermediates (ROI) release by peritoneal exudate cells (PEC) and histopathological findings in the prostate gland were assessed during experimental autoimmune prostatitis (EAP) development. Results in EAP rats were compared with data from rats immunized with kidney homogenate, BSA, and CFA, as well as nontreated rats. At 28 days of first immunization (FI), EAP rats spontaneously released significantly more ROI than occurred in the cells from control rats. A similar response was found when ROI release was analyzed after in vitro stimulus. In time course studies, an increased spontaneous O2- production was observed at day 7 after FI, and remained the same during all period studied, (14, 21, and 28 days after FI). The stimulated O2- production showed elevated levels at 7 days after FI and fell afterward to levels similar to those of nontreated rats and increased again at 28 days. Spontaneous or stimulated H2O2 release showed a progressive increase during the study periods. ROI release was correlated with infiltrate formation in the prostate gland. This differential responsiveness could indicate that, during the autoimmune process, the autoantigen(s) amplify the inflammatory response triggered by them.

In vitro study of human alveolar macrophage and peripheral blood mononuclear cell reactive oxygen-intermediates release induced by sulfur dioxide at different concentrations.

Sulfur dioxide (SO2) is a major air pollutant in urban areas. Alveolar macrophages (AM) located on the alveolar surface are in direct contact with this inhaled gas. We evaluated the dose-dependent effect of SO2 exposure on the oxidative metabolism of AM and peripheral blood mononuclear cells (PBMNC) by measuring the spontaneous and stimulated reactive oxygen intermediates (ROI) release. AM or PBMNC were placed on a polycarbonate membrane, which was in direct contact with the surface of a nutrient reservoir. For exposure of the cells to SO2 a special chamber was employed, in which humidified standard air with 5% CO2 at 37 degrees C was mixed with SO2 at the desired concentration. Periods of time between 30 and 120 minutes and concentrations between 0.3 and 1.5 ppm SO2 were chosen for exposure. Thirty minutes exposure of AM to SO2 (0.3-1.5 ppm) yielded a dose-dependent stimulation of ROI release; 2.0- to 3.6-fold of control (r = 0.965, p < 0.005). An exposure of 120 minutes to SO2 resulted in a similar ROI production of about 2.5-fold at all tested concentrations. These experiments provide evidence that AM and PBMNC become activated by SO2 producing large amounts of ROI.

Different influences of cytochalasin B on the activation of human neurophils settled onto petri dishes displayed by simultaneously detected native and luminol‐dependent luminescence

Cytochalasin B (CB) is known to interfere reversibly with the cytoplasmic contractile filamental network of mammalian cells. The role of the microfilament system in the mechanism of the reactive oxygen intermediates release of polymorphonuclear leukocytes (PMNL) was studied for different kinds of stimuli. PMNL from fresh human blood were treated with CB and stimulated by adherence on plastic surfaces, by opsonized zymosan, by phorbol myristate acetate and by N-formylmethionyl-phenylalaline. The production of reactive oxygen species were monitored by simultaneous detection of native, luminol-independent, luminescence (NL) and luminol-dependent luminescence (LDL) using a method of spectral discrimination. Different influences of CB on NL with respect to LDL as well stimuli-dependent influences of CB on the luminescence response of PMNL were observed. Especially phagocytosis-associated activation of PMNL was strongly inhibited by CB, whereas LDL was reduced to a much greater extent in comparison with NL. A firm involvement of the microfilament system is indicated, but it depends on the kind of stimulus engaged.

Correlation between release of reactive oxygen intermediates and inhibition of toxoplasma multiplication in mouse peritoneal and alveolar macrophages and kidney cells after in vitro incubation with obioactin, lonomycin a, muramyl dipeptide, lipopolysaccharide or toxoplasma lysate antigen

The inhibition of Toxoplasma multiplication inside cells does not correlate with an enhanced release of oxygen intermediates except in the case of peritoneal macrophages treated with Obioactin. The inhibition observed in alveolar macrophages treated with Obioactin, in kidney cells treated with Obioactin or lonomycin A and in peritoneal macrophages treated with lonomycin A was not accompanied by an increment of release of oxygen intermediates. Lipopolysaccharide (LPS) and muramyl dipeptide (MDP) enhanced the release of toxic oxygen intermediates in peritoneal macrophages, but did not have any toxoplasmacidal effect. Adenosine triphosphate (ATP) content increased during Obioactin, MDP or Toxoplasma lysate antigen (TLA) treatment. The actual oxygen consumption of the peritoneal macrophages treated with Obioactin increased dose dependently, but that of TLA-, lonomycin A- or MDP-treated cells did not change. These results suggest that the relationship between the intracellular killing of Toxoplasma protozoa and the release of oxygen intermediates differs according to the cells and/or the stimuli, and that the cellular mechanism of Toxoplasma killing in the peritoneal macrophages treated with Obioactin involves an energy-dependent mechanism.

Biological Activity of &lt;italic&gt;Pityrosporum&lt;/italic&gt;. II. Antitumor and Immune Stimulating Effect of &lt;italic&gt;Pityrosporum&lt;/italic&gt; in Mice

The antitumor activity of Pityrosporum (P. orbiculare, P. ovale, P. pachydermatis, and Pityrosporum sp.) on Ehrlich ascites carcinomas (EACs) implanted into outbred ICR mice was studied. Pityrosporum significantly prolonged the survival of mice, regardless of the administration mode. In the case of P. orbiculare, the maximum survival time was 32.3 days on the mean and was obtained by injection ip of 1 mg (dry weight) P. orbiculare for 7 consecutive days following inoculation of the tumor cells. In contrast, the mean survival time of the nontreated mice was 14.9 days. For the investigation of the mechanisms of this antitumor activity, an examination was done on the ability of intracellular killing of Salmonella typhimurium and oxygen intermediate release by Pityrosporum, as elicited by mouse peritoneal exudate cells (PEC) or mouse peritoneal macrophages (PM). With about 40-minute incubation, 60-80% of S. typhimurium phagocytized by Pityrosporum elicited PEC or PM or were killed. The amounts of superoxide released from Pityrosporum-elicited cells were 1.5 times higher than those of P. acnes-elicited ones. Furthermore, three serum proteins (LA, LB, and LC), which closely related to the anti-tumor activity of immunomodulators, increased in the mice given Pityrosporum. These results indicated that the better survival rate seen in the case of Pityrosporum administration to mice with an implanted EAC may relate to the potent activation of phagocytes and to the increase in serum proteins LA, LB, and LC.

Serum factor requirement for reactive oxygen intermediate release by rabbit alveolar macrophages.

Alveolar macrophages (AM) from pathogen-free rabbits were unable to release reactive oxygen intermediates (ROI) unless they were conditioned in serum for 24-48 h before triggering with membrane-active agents. The degree of serum conditioning of AM depended upon the concentration of serum used; optimal ROI release was obtained at or above 7.5% fetal bovine serum (FBS). FBS, autologous rabbit serum, pooled rabbit serum, and pooled human serum were each capable of conditioning AM for release of ROI. Serum conditioning of AM requires synthesis of new protein(s); and the enzyme required for ROI production, NADPH oxidase, was only detectable in serum-conditioned cells. Moreover, serum-conditioned cells lost their ability to release ROI after transfer to serum-free medium, while cells maintained in serum-free medium acquired the capacity to release ROI after their transfer to serum-containing medium, demonstrating the reversibility of the phenomenon. Initial purification data indicate that conditioning is mediated by a discrete serum constituent, which precipitates 40-80% saturated ammonium sulfate, does not bind to Cibacron Blue columns, and has a molecular weight of 30,000 to 50,000, as determined by molecular exclusion chromatography. Unlike gamma interferon, which also enhances ROI release by macrophages, our serum-conditioning factor is not acid labile, retaining 67% of its activity after 120 min incubation at pH 2.0. Moreover, it does not appear to be a contaminating endotoxin, since LPS neither conditioned AM for ROI production, nor triggered ROI production by serum-conditioned AM. We propose that such a conditioning requirement may normally protect the lung against ROI-mediated tissue injury. However, during a pulmonary inflammatory reaction initiated by other mediator systems, the resulting transudation of plasma proteins into the alveolar spaces may condition AM in situ for ROI production.