Accumulation of Cytochrome b558 at the Plasma Membrane: Hallmark of Oxidative Stress in Phagocytic Cells.

Neutrophils play a very key role in the human immune defense against pathogenic infections. The predominant players in this role during the activation of neutrophils are the release of cytotoxic agents stored in the granules and secretory vesicles and the massive production of reactive oxygen species (ROS) initiated by the enzyme NADPH oxidase. In addition, in living organisms, cells are continuously exposed to endogenous (inflammations, elevated neutrophil presence in the vicinity) and exogenous ROS at low and moderate levels (travels by plane, radiotherapy, space irradiation, blood banking, etc.). To study these effects, we used ROS induced by gamma radiation from low (0.2 Gy) to high (25 Gy) dose levels on PLB-985 cells from a myeloid cell line differentiated to neutrophil-like cells that are considered a good alternative to neutrophils. We determined a much longer lifetime of PLB-985 cells than that of neutrophils, which, as expected, decreased by increasing the irradiation dose. In the absence of any secondary stimulus, a very low production of ROS is detected with no significant difference between irradiated and non-irradiated cells. However, in phagocytosing cells, irradiation doses above 2 Gy enhanced oxidative burst in PLB-985 cells. Whatever the irradiation dose, NADPH oxidase devoid of its cytosolic regulatory units is observed at the plasma membrane in irradiated PLB-985 cells. This result is different from that observed for irradiated neutrophils in which irradiation also induced a translocation of regulatory subunits suggesting that the signal transduction mechanism or pathway operate differently in both cells.

Modulation of cytokine and nitric oxide production by keratinocytes, epithelial cells, and mononuclear phagocytes in a co-culture model of inflammatory acne.

INTRODUCTION: Ultraviolet B (UVB, 290 nm to 320 nm) has been reported to modulate the cytokine-mediated inflammatory process in various inflammatory skin conditions, including production of TNF-α, IL-1α, IL-6, IL-8, and IL-10. We constructed an in vitro model system involving co-culture of different cell types to study the effect of UVB on the inflammatory process using nitric oxide (NO) and tumor necrosis factor (TNF)-α as markers of inflammation. OBJECTIVE: This study was conducted to quantitatively assess the products secreted by human epithelial keratinocytes in the presence and absence of macrophages/monocytes. METHODS: Cells were exposed to UVB radiation (50 mJ to 200 mJ per cm2) or treated with bacterial lipopolysaccharide (LPS) as stimulator of inflammatory response. Nitric oxide (NO) was measured by modified Griess assay and TNF-α was measured by quantitative ELISA. For the co-culture system, SC monocytes were seeded in a 24-well Transwell tissue culture plate whereas irradiated keratinocytes were seeded in the individual baskets subsequently placed on top of the monocyte cultures, and samples of culture supernatants were collected at 1 to 6 days. RESULTS: When primary human epidermal keratinocytes (NHEK) were irradiated with UVB, a dose-dependent stimulation of TNF-α production was observed (33% to 200% increase). TNF-α production was not changed significantly in SC monocytes/NHEK co-culture. In contrast, when macrophages were irradiated with UVB, significant inhibition of NO production (40% suppression, P<0.001) was seen. CONCLUSION: This improved model of cutaneous inflammation could use multiple cells to study their interactions and to offer convenience, reproducibility, and a closer approximation of in vivo conditions.

Phagocyte functions of human subjects living in high level of natural radiation areas in Iran.

BACKGROUND: Iran is considered as one of the countries which are located in high radiation area. Ramssar contains natural radioactive sources (Uranium-238) that can produce Radium-226 with high solubility and Radon gas with ability to fuse in dirt, soil, and water in its decay chain. Radioactive rays can produce free radicals with a short life time but high energy, which lead to changes structures of protein bindings, hormones activity, lipids oxidation and DNA breakage which can induce autoimmune diseases and other disorders. The aim of this study was to evaluate effects of natural radiation on innate and humoral immune response of those exposed to natural radioactive rays compared to control group. METHODS: Subjects from high and low natural radiation areas were included in the study. Neutrophil chemotaxis, Nitro-Blue Tetrazolium (NBT), and antioxidant effects were evaluated for effects of radioactivity on free radicals and innate immunity. Also Cytokines (IL-2, IL-4) levels for humoral immune system of the subjects were measured. RESULTS: Total plasma antioxidant level and potency of respiratory burst in individuals who lived in area with high levels of natural radiation decreased significantly compared with, control subjects who lived in the other areas of Ramssar with normal radiation. The level of IL-4 increased in individuals who lived in area with high levels of natural radiation, which could lead to Th2 pattern of immune response whether neutrophils chemotaxis in the groups living in the area with high level of natural radiation didn't change significantly when compared to control. CONCLUSION: Neutrophil respiratory burst activities suggested that NADPH probably reduced due to different reasons including decrease in NADPH oxidase and other enzymes, and mutation of the genes or production of oxidant substances. Neutrophil chemotaxis reduced but this reduction was not statistically significant.

HClO-Activated Fluorescence and Photosensitization from an AIE Nanoprobe for Image-Guided Bacterial Ablation in Phagocytes.

Bacteria hiding in host phagocytes are difficult to kill, which can cause phagocyte disorders resulting in local and systemic tissue damage. Effective accumulation of activatable photosensitizers (PSs) in phagocytes to realize selective imaging and on-demand photodynamic ablation of bacteria is of great scientific and practical interests for precise bacteria diagnosis and treatment. Herein, HClO-activatable theranostic nanoprobes, DTF-FFP NPs, for image-guided bacterial ablation in phagocytes are introduced. DTF-FFP NPs are prepared by nanoprecipitation of an HClO-responsive near-infrared molecule FFP and an efficient PS DTF with aggregation-induced emission characteristic using an amphiphilic polymer Pluronic F127 as the encapsulation matrix. As an energy acceptor, FFP can quench both fluorescence and production of reactive oxygen species (ROS) of DTF, thus eliminating the phototoxicity of DTF-FFP NPs in normal cells and tissues. Once delivered to the infection sites, DTF-FFP NPs light up with red fluorescence and efficiently generate ROS owing to the degradation of FFP by the stimulated release of HClO in phagocytes. The selective activation of fluorescence and photosensitization is successfully confirmed by both in vitro and in vivo results, demonstrating the effectiveness and theranostic potential of DTF-FFP NPs in precise bacterial therapy.

Long-term UVB irradiation affects the immune functions of carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

The effects of long-term, low-dose ultraviolet B (UVB) radiation on immune functions of two fish species representing different taxonomic groups, carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss), were assessed in this study. The fish were exposed to 7, 20 or 60 mJ cm(-2) UVB three times per week, for 6 weeks. In carp, UVB exposure affected the respiratory burst activity of blood and head kidney phagocytes, differential blood leukocyte counts and blood chemistry. Phytohemagglutinin (PHA)-stimulated in vitro proliferation responses of blood and head kidney lymphocytes, however, remained unchanged. Rainbow trout tolerated the irradiations with fewer alterations, but significant changes were detected in blood chemistry and hematocrits of the irradiated fish. These results indicate that long-term exposure to low doses of UVB induces immunomodulation in fish, and that there are species-specific differences in sensitivity to irradiation.

Phagocytic response of astrocytes to damaged neighboring cells.

This study aims to understand the phagocytic response of astrocytes to the injury of neurons or other astrocytes at the single cell level. Laser nanosurgery was used to damage individual cells in both primary mouse cortical astrocytes and an established astrocyte cell line. In both cases, the release of material/substances from laser-irradiated astrocytes or neurons induced a phagocytic response in near-by astrocytes. Propidium iodide stained DNA originating from irradiated cells was visible in vesicles of neighboring cells, confirming phagocytosis of material from damaged cortical cells. In the presence of an intracellular pH indicator dye, newly formed vesicles correspond to acidic pH fluorescence, thus suggesting lysosome bound degradation of cellular debris. Cells with shared membrane connections prior to laser damage had a significantly higher frequency of induced phagocytosis compared to isolated cells with no shared membrane. The increase in phagocytic response of cells with a shared membrane occurred regardless of the extent of shared membrane (a thin filopodial connection vs. a cell cluster with significant shared membrane). In addition to the presence (or lack) of a membrane connection, variation in phagocytic ability was also observed with differences in injury location within the cell and distance separating isolated astrocytes. These results demonstrate the ability of an astrocyte to respond to the damage of a single cell, be it another astrocyte, or a neuron. This single-cell level of analysis results in a better understanding of the role of astrocytes to maintain homeostasis in the CNS, particularly in the sensing and removal of debris in damaged or pathologic nervous tissue.

Radiation therapy primes tumors for nanotherapeutic delivery via macrophage-mediated vascular bursts.

Efficient delivery of therapeutic nanoparticles (TNPs) to tumors is critical in improving efficacy, yet strategies that universally maximize tumoral targeting by TNP modification have been difficult to achieve in the clinic. Instead of focusing on TNP optimization, we show that the tumor microenvironment itself can be therapeutically primed to facilitate accumulation of multiple clinically relevant TNPs. Building on the recent finding that tumor-associated macrophages (TAM) can serve as nanoparticle drug depots, we demonstrate that local tumor irradiation substantially increases TAM relative to tumor cells and, thus, TNP delivery. High-resolution intravital imaging reveals that after radiation, TAM primarily accumulate adjacent to microvasculature, elicit dynamic bursts of extravasation, and subsequently enhance drug uptake in neighboring tumor cells. TAM depletion eliminates otherwise beneficial radiation effects on TNP accumulation and efficacy, and controls with unencapsulated drug show that radiation effects are more pronounced with TNPs. Priming with combined radiation and cyclophosphamide enhances vascular bursting and tumoral TNP concentration, in some cases leading to a sixfold increase of TNP accumulation in the tumor, reaching 6% of the injected dose per gram of tissue. Radiation therapy alters tumors for enhanced TNP delivery in a TAM-dependent fashion, and these observations have implications for the design of next-generation tumor-targeted nanomaterials and clinical trials for adjuvant strategies.

The immunological effects of extracorporeal photopheresis unraveled: induction of tolerogenic dendritic cells in vitro and regulatory T cells in vivo.

Extracorporeal photopheresis (ECP) may represent an alternative to immunosuppression, as a means of reducing rejection after thoracic organ transplantation. The mechanism by which ECP exerts its protective effects has, until now, remained elusive. We analyzed peripheral blood mononuclear cells of four children with chronic heart and lung transplant rejection, who received ECP in addition to conventional immunosuppressive treatment. The effects of ECP were evaluated at each cycle, comparing blood samples from the same patient collected before and after treatment. In vitro, peripheral blood mononuclear cells treated with ECP undergo apoptosis and are phagocytosed by immature dendritic cells, which, in turn, acquire a tolerogenic phenotype. The frequency of T cells, with a regulatory phenotype and strong suppressive activity, was significantly increased in the blood of ECP-treated patients. The immunomodulatory effects of ECP may be explained by its ability to increase the frequency of regulatory T cells with inhibitory action on transplant immune rejection.

Time course and role of the pineal gland in photoperiod control of innate immune cell functions in male Siberian hamsters.

The time course of select phagocyte and natural killer activities to short days was determined. In advance of testes regression, circulating granulocyte and monocyte cell numbers in hamsters decreased while lymphocyte numbers increased; phagocytosis and oxidative burst activity also decreased. To determine whether the pineal gland influences these innate immune cell functions, hamsters were exposed to constant light. Photoperiod control of testes weight and basal oxidative burst activity was abolished by treatment with constant light; other phagocyte activities and leukocyte proportions in circulation were not affected. The findings suggest that photoperiod and pineal gland function may regulate certain innate immune activities.

Ultraviolet B irradiation modulates the immune system of fish (Rutilus rutilus, Cyprinidae). I. Phagocytes.

Roach (Rutilus rutilus) were irradiated with a single dose of ultraviolet B (UVB) radiation (0.4 J/cm2) in order to study the effects of UVB on the nonspecific immune defense mechanisms of fish. Neutrophils and macrophages were isolated from the head kidney of fish on days 1-14 postirradiation. Both random and directed migration of neutrophils, studied by migration under agarose assay, were suppressed on day 1 after UVB irradiation. The respiratory burst of phorbol 12-myristate 13-acetate-stimulated neutrophils and macrophages was also suppressed at days 1 and 2 after UVB irradiation. The suppression of migration and respiratory burst were restored or the responses were even enhanced later, but on the other hand spontaneous cytotoxicity of neutrophils toward 51chromium-labeled K562 target cells stayed suppressed throughout the 14 day follow-up. This study indicates that UVB radiation has the potential to suppress the functioning of phagocytes and to compromise the immune system of fish.

Impairment of phagocytic cell respiratory burst by UVA in the presence of fluoroquinolones: an oxygen-dependent phototoxic damage to cell surface microvilli.

Fluoroquinolones are widely used clinically as broad-spectrum antimicrobial agents. One of their side effects is UVA-dependent photosensitivity, observed after the skin is exposed to sunlight. We have investigated five fluoroquinolones and have found that their phototoxicity is oxygen dependent. Human phagocytic leucocytes were stimulated with serum opsonized zymosan to produce superoxide radical (O2-) (respiratory burst) in the presence of a sensitive O2(-)-specific cypridina luciferin analogue, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazol (1,2-alpha) pyrazin-one hydrochloride (MCLA), as chemiluminescence reagent with which O2- can react to induce photon emission. The photon count was used as a measure of respiratory burst activity. When leucocytes were irradiated with UVA for 10 min in the presence of 3 micrograms ml-1 lomefloxacin, ciprofloxacin or norfloxacin, a marked decrease in respiratory burst activity was observed; in this respect, ofloxacin and tosufloxacin were weak. Scanning electron microscopy revealed that the cell surface microvilli were destroyed. The phototoxicity of fluoroquinolones could be abolished if oxygen in the tests was replaced by nitrogen or if the aminothiol DL-cysteine (1.5 mg ml-1) was added prior to irradiation. It is suggested that an oxygen species derived from UVA-excited drug molecules and oxygen mediates the phototoxicity of these fluoroquinolones.

The protective activity of Thymex L against radiotherapeutically-induced cellular immunodepression in lung cancer patients.

In order to prevent the radiotherapeutically-induced aggravation of initial immunodeficiency, a thymic preparation (Thymex L) was given to lung cancer patients simultaneously with irradiation. The parameters of both cellular and humoral nonspecific immunity were evaluated in two groups of patients: one was treated with radiotherapy only (60 Gy in 30 fractions); the other one received Thymex L (100 mg 3 times a week, total dose 1800 mg, i.m.) simultaneously with radiotherapy. The significant decrease of B and T cell number, and decreased lymphoproliferative response to PHA were found in all patients before therapy; the number and phagocyting capacity of blood monocytes, as well as the concentrations of circulating IgG, IgA and immunocomplexes, were all significantly increased. Immediately after irradiation the patients had even lower number of T and B cells, diminished reactivity to PHA and higher number of mononuclear phagocytes when compared to the values before therapy. In patients treated with Thymex L, the number of B and T cells and PHA-induced proliferative response were significantly higher than in those treated with radiotherapy only. No effect of this therapy was seen on active T cells, on high number and function of mononuclear phagocytes and on elevated concentrations of serum immunoglobulins and immune complexes. Our results indicate that Thymex L can successfully prevent the harmful effect of radiation therapy on cellular immunity in a majority of lung cancer patients.

[Phagocytic lymphocyte-like elements (PLLE) and irradiation in vitro]. / Elementi simil-linfocitari fagocitanti (E.S.L.F.) e irradiazione in vitro.

The peripheral blood of rats contains lymphocyte-like phagocytes after intracardial injection of India ink. In vitro irradiation of whole blood (1000 r - 60Co), causes marked reduction in whole lymphocytes but does not modify the number and morphological integrity of the phagocytes, which can therefore be held to be comparatively radioresistant: as such, they could belong either to the mononucleate phagocyte system or to the lymphatic system (T4 lymphocytes). Since T-lymphocytes are not capable of phagocytosis, the former hypothesis would appear the most probable.

Role of phagocytes in antimicrobial defence of the middle ear.

The role of phagocytes in the antimicrobial defence of the middle ear was investigated in this experiment, using Hartley strain guinea pigs with an experimental otitis media. Otitis media was induced with an inoculation of Streptococcus pneumoniae into the tympanic cavity through the ear drum. For depletion of peripheral blood phagocyte population such as monocytes and polymorphonuclear neutrophils (PMNs), whole body irradiation (250 rad or 500 rad) was carried out on guinea pigs three days before S. pneumoniae inoculation into the middle ears. Carrageenan was also used for selective depletion of mononuclear cells, to distinguish their role from polymorphonuclear neutrophils. In control animals, otitis media was induced reproducibly with middle ear inoculation of more than 10(6) S. pneumoniae. In irradiated animals, which underwent 10(2) or 10(4) S. pneumoniae inoculation, the incidence of otitis media because of S. pneumoniae infection became higher in accordance with the dosage of irradiation. However, no significant difference was seen in the occurrence of otitis media and the number of viable bacteria recovered from bulla washings between controls and carrageenan-treated animals. These results suggest that phagocytes, particularly neutrophils, are essential for antimicrobial defense at the early phase of the middle ear infection with S. pneumoniae.

[Bioeffects of chronic exposure to radiofrequency electromagnetic fields of low intensity (standardization strategy)]. / Bioéffekty khronicheskogo vozdeistviia élektromagnitnykh polei radiochastotnogo diapazona malykh intensivnostei (strategiia normirovaniia).

A retrospective analysis of the experimental researches on the effect of radio frequency electromagnetic fields (EMF) on human health, carried out in the USSR, is presented. The results of chronic exposure of laboratory animals to EMF have been considered. Apparently, EMF in the range of 1750-2750 MHz with power density up to 100-500 W/cm2 caused in immune globullin fractions, and a development of autoimmune processes. The changes in parameters of reproductive functions and posterity, the increase in embryo mortality were found. The standartization strategy used in the USSR and currently applied in Russia has been discussed.

[Molecular and cellular mechanisms of the low intensity laser radiation effect]. / Molekuliarno-kletochnye mekhanizmy deistviia nizkointensivnogo lazernogo izlucheniia.

The main aspects of the free radical conception of the molecular and cellular mechanisms of the stimulating action of low-intensity radiation in the red region of the spectrum were considered. These are: (1) Primary acceptors of incident radiation are endogenous porphyrins, which may act as photosensitizers giving initiator-radicals for secondary free radical reactions. (2) Target cells for light irradiation during quantum therapy may be blood leukocytes, fibroblasts, keratinocytes, endotheliocytes, etc. (3) The initiation of the secondary free radical reactions due to lipid peroxidation of cell membranes (in particular, of leukocytes) brings about an increase in ion permeability including that for calcium. The increase in intracellular calcium concentration leads to phagocytes priming, i.e., to increased production of reactive oxygen species (ROS) under subsequent stimulation of the cell. (4) Photosensitized generation of ROS in the cytoplasm of some cells induces a free-radical activation of synthesis of proteins, the most significant in the light of the present concept being the de novo synthesis of inducible NO-synthase, superoxide dismutase, and various cytokines. The experimental evidence for the basic statements of the conception of free radical mechanisms for the stimulating action of low-intensity laser and noncoherent radiations is presented. A relation between the primary mechanisms of the stimulating action of light and the secondary effects that determine the sanative effect of quantum therapy in the process of wound healing (bactericidity, cell proliferation, and improved microcirculation) was established. Moreover, it was shown that nitrosyl complexes of heme proteins, such as hemoglobin and cytochrome c, are the primary chromophores of laser radiation. Upon irradiation, they can easily dissociate to produce free nitric oxide. In turn, released nitric oxide may be responsible for blood vessel relaxation and activation of mitochondrial respiration. This phenomenon is just observed during phototherapy by means of low-intensity laser radiation.

The impact of mouse strain on iron ion radio-immune response of leukocyte populations.

PURPOSE: Exposure to various forms of radiation, including iron ions that have an exceptionally high biological effectiveness, is an inevitable consequence of spaceflight. However, genetic background can significantly influence the response to radiation and hence also the overall health of crewmembers. The major goal of this study was to compare leukocyte population responses in two strains of mice that differ in susceptibility to radiation: C57BL/6 (resistant) and CBA/Ca (susceptible). MATERIALS AND METHODS: The mice were whole-body irradiated with 0, 50, 200, or 300 cGy (56)Fe(26) (1 GeV) at approximately 1 Gy/min and euthanised on days 4 and 30 thereafter for analyses. Analyses included body and organ masses (spleen, liver, thymus, lungs), distribution of leukocyte populations in blood and spleen, red blood cell and platelet characteristics, expression of surface molecules (CD11b, CD54), and spontaneous and mitogen-induced blastogenesis. RESULTS: There were main effects of Dose and Dose x Day interactions on virtually all quantified parameters in both strains of mice. In contrast, there were relatively few Dose x Strain and three-way interactions. Strain-related interactions involved changes in circulating phagocytic populations, erythrocytes, and liver mass. CONCLUSION: The data demonstrate that genetic background can modify certain immune-related parameters after exposure to heavy particle radiation. The possible implications of these findings are discussed.