The role of lipid peroxidation products in the effect of He-Ne laser on human blood leukocytes

The role of lipid peroxidation products formed in membranes of human blood leukocytes after irradiation with He-Ne laser was studied. It was found that low-intensity laser irradiation (0.3-1.6 J/cm2) leads to both cell activation and an increase in the content of lipid peroxidation products. The intensity of lipid peroxidation was analyzed by estimating the amount of TBA reactive products and lipid diene conjugates. Irradiation in the presence of an exogenous photosensitizer (protoporphyrin IX) enhanced the phenomena observed. The use of antioxidants (tocopherol and ionol) completely eliminated the laser-induced effects (changes in leukocyte activity and accumulation of lipid peroxidation products). These results can be explained by the fact that laser irradiation leads to the activation of lipid peroxidation in leukocyte membranes, which in turn enhances the response of cells to the stimulus (priming).     

The role of blood plasma porphyrins in the effects of He-Ne-laser on human leukocytes

The He-Ne-laser induced effects in human blood leukocytes in the presence of autologic plasma were investigated. Experiments were performed in two ways: (1) He-Ne-laser irradiation of cells in the presence of autologic plasma or (2) laser irradiation and subsequent addition of autologic plasma to the cell suspension. The concentration dependencies of plasma additions were evaluated. To obtain different concentrations of porphyrins in plasma samples, we either diluted the samples with PBS or selected patients with different porphyrin plasma content. The effects of He-Ne-laser irradiation were characterized by the maximum effect dose (Dmax) of irradiation and the degree of maximum cell activation (Amax, priming index). In the first series of experiments, we irradiated leukocytes in the presence of autologic plasma taken from patients with pneumonia and bronchial asthma. It was found that Dmax decreased with increasing porphyrin concentration in plasma. It was observed that, at low porphyrin concentrations, Amax increased severalfold with increasing photosensitizer concentration. At a porphyrin concentration of 0.46 pmol a decrease in Amax was detected as the porhyrin concentration increased. The same effects were revealed at high doses of laser irradiation. Very similar effects were found in experiments with the addition of irradiated plasma to cells. However, the Amax value was considerably less compared to that after irradiation in the presence of plasma (160% vs. 230 - 270% upon combined irradiation of cells and plasma). The Dmax value was higher in the series of experiments in which plasma was irradiated separately. The results suggest that laser-induced leukocyte activation can be mediated by blood plasma porphyrins and the products of lipid peroxidation formed as a result of porphyrin-photosensitized lipid oxidation.     

Mitochondria, endoplasmic reticulum and actin filament behavior after PDT with chloroaluminum phthalocyanine liposomal in HeLa cells

Photodynamic therapy (PDT) for cancer is a therapeutic modality in the treatment of tumors in which visible light is used to activate a photosensitizer. Cell membranes have been identified as an important intracellular target for singlet oxygen produced during the photochemical pathway. This study analyzed the cytotoxicity in specific cellular targets of a photosensitizer used in PDT in vitro. The photosensitizing effects of chloroaluminum phthalocyanine liposomal were studied on the mitochondria, cytoskeleton and endoplasmic reticulum of HeLa cells. Cells were irradiated with a diode laser working at 670 nm, energy density of 4.5 J/cm2 and power density of 45 mW/cm2. Fluorescence microscopic analysis of the mitochondria showed changes in membrane potential. After PDT treatment, the cytoskeleton and endoplasmic reticulum presented basic alterations in distribution. The combined effect of AlPHCl liposomal and red light in the HeLa cell line induced photodamage to the mitochondria, endoplasmic reticulum and actin filaments in the cytoskeleton.     

Inhibitory and enhancing effects of piroxicam on whole blood chemiluminescence.

The effects of piroxicam on the production of reactive oxygen species by stimulated phagocytes was studied in whole blood by a chemiluminescence (CL) technique in relation to maximum activity, localization and kinetics of radical generation. We found that piroxicam dose-dependently inhibited total (intra- and extracellular) zymosan-stimulated luminol CL (LCL) at a high stimulant concentration (p = 0.0001). Piroxicam additionally decreased cytochalasin B-reduced LCL, which shows that the effect of the drug should be sought in the extracellular component of the response. Piroxicam inhibited the first phase of extracellular LCL in a dose-dependent manner (p = 0.0001) and revealed itself as an enhancing agent of CL in later time intervals after the start of respiratory burst, in a model system containing horseradish peroxidase (HRP) and sodium azide. It enhanced LCL of a cell-free system, i.e. influenced the CL due to HRP-catalysed decomposition of hydrogen peroxide. It also dose-dependently inhibited the early extracellular superoxide production, evaluated by lucigenin CL (p = 0.022). Piroxicam inhibited the total fMLP-stimulated LCL by 70% approximately and, only by about 30%, the first phase of fMLP-stimulated extracellular LCL, which presupposes an effect on myeloperoxidase-catalysed formation of hypochloric acid. Piroxicam slightly increased the intracellular LCL by phagocytes (p = 0.02), an effect that is probably connected with its ability to induce the release of secondary messengers in signal transduction. In conclusion, the anti-inflammatory effect of piroxicam is probably related to the inhibition of the extracellular generation of superoxide and hypochloric acid in the early stages of phagocyte activation.     

Immunomodulation by exogenous surfactant: effect on TNF-alpha secretion and luminol-enhanced chemiluminescence activity by murine macrophages stimulated with group B streptococci

Group B streptococci (GBS) are important pathogens in neonatal sepsis and pneumonia. GBS stimulate alveolar macrophages to produce inflammatory cytokines and free oxygen radicals, which can damage the lungs. In several studies, use of exogenous surfactant in term babies has improved outcome related to sepsis and respiratory failure. The role(s) of exogenous surfactant in modulating the inflammatory response produced by this microbe was examined. Tumor necrosis factor alpha (TNF-alpha) production and luminol-enhanced chemiluminescence (LCL), a measure of respiratory burst, were investigated. For measuring TNF-alpha release, RAW 264.7 murine macrophages were pre-incubated with bovine surfactant and stimulated with either lipopolysaccharide, live or heat-killed GBS type Ia. LCL was measured after macrophages were pre-incubated with or without surfactant overnight, then stimulated with GBS or phorbol myristate acetate. Lipopolysaccharide and GBS stimulated TNF-alpha secretion from macrophages that was suppressed by exogenous surfactant in a dose-dependent fashion. GBS and phorbol myristate acetate also increased LCL from macrophages, which was significantly suppressed by pre-incubation of macrophages with exogenous surfactant. We conclude that GBS type Ia stimulates TNF-alpha release and LCL from RAW 264.7 cells and that these responses are suppressed by surfactant. Suppression of inflammatory mediators by exogenous surfactant might improve respiratory disease associated with GBS.     

Priming activity for chemiluminescence reaction of PMN in the culture supernatant of streptococcal preparation (OK-432)-stimulated spleen cells

We investigated the effect of supernatant from human spleen cell culture stimulated with a streptococcal preparation, OK-432 (OK sup), on the luminol-dependent chemiluminescence (CL) of human polymorphonuclear leukocytes (PMN). The fMLP-stimulated CL of PMN was markedly enhanced by the pretreatment with OK sup. This result indicates that OK sup contained the factor(s) that primes fMLP-stimulated CL of PMN. The priming factor(s) in OK sup was partially inactivated by the treatment at 56 C for 30 min and pH 2 or pH 10 treatments. Since the enhancing effect of OK sup on the CL was inhibited by the treatment of sodium azide and the addition of catalase or taurine, it was assumed that OK sup augments the activity of MPO-H2O2-HOCl system of fMLP-stimulated PMN.     

Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen

Characterization of protein damage during photosensitization of chlorin e6-treated cells was performed using the green fluorescent protein (GFP). The GFP-chromophore damage caused by singlet oxygen was studied in COS 7 kidney cells and E. coli bacteria following light irradiation. Electron spin resonance (ESR) revealed the generation of endogenous singlet oxygen (1O2) by photoactivated GFP, an effect similar to that produced by the exogenous photosensitizer chlorin e6. A light dose-dependent photobleaching effect of GFP was pronounced at low pH or upon photosensitization with chlorin e6. However, the 1O2 quenchers beta-carotene and sodium azide minimized GFP photo-bleaching. Gel electrophoresis of photosensitized GFP followed by fluorescence multi-pixel spectral imaging revealed the binding of chlorin e6 to GFP, affecting the photobleaching efficacy. Fluorescence multi-pixel spectral imaging of GFP-transfected COS 7 cells demonstrated the presence of GFP in the cytoplasm and nucleus, while chlorin e6 was found to be concentrated in the perinuclear vesicles. Exposure of the cells to light induced GFP photobleaching in the close vicinity of chlorin e6 vesicles. We conclude that photoactivated GFP generates endogenous 1O2, inducing chromophore damage, which can be enhanced by the cooperation of exogenous chlorin e6.     

Effect of oxidized phospholipids on the chemiluminescence of zymosan-activated leukocytes

The effect of liposomes with different degree of oxidation on the zymosan-induced chemiluminescence (CL) of leukocytes was investigated. Non-oxidized liposomes did not influence significantly the CL response of leukocytes. In contrast previously oxidized liposomes increased CL even if liposomes and cells were separated by a dialysis membrane. Based on the observed increase of luminol-activated CL by oxidized liposomes, lipid peroxidation (LPO) products may be suggested to enhance cell activation. Zymosan-activated leukocytes did not affect the amount of malondialdehyde (MDA) in non-oxidized liposomes unless iron salts were added. Fe3+ + ADP added to non-oxidized liposomes triggered LPO. Both catalase and superoxide dismutase (SOD) prevented the effect. In experiments with previously oxidized liposomes the activated oxygen species produced by leukocytes did not increase the amount of MDA; on the contrary, they decreased it both in the presence and in the absence of chelated iron in the liposome suspension. The reaction between lipid hydroperoxide and O2- widely accompanied by CL. SOD decreased CL in this system by a factor of 1.7. On the other hand, peroxidized lipids may "opsonize" initially inactive particles: oxidized liposomes increased CL response of leukocytes similarly as opsonized zymosan routinely used as a phagocyte activator.     

Chemiluminescence response of beta-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax

The respiratory burst of leukocytes isolated from sea bass (Dicentrarchus labrax) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with beta-glucan. The CL response by P.C. and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood. This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros. Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros. Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction). Scanning electron microscopy observations were also carried out.Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O-2 dismutation to H(2)O(2), supported the correlation between CL and respiratory burst. Treatment with ouabain and DNP suggested that in this response, Ca(++) pump channels and calmodulin are involved in a metabolic energy-dependent pathway.     

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.     

Isolation of human salivary polymorphonuclear leukocytes and their stimulation-coupled responses.

A simple method was developed to isolate viable human salivary polymorphonuclear leukocytes (SPMN) from the oral cavity, and stimulation-coupled responses of these cells were examined. From morphological characteristics and the presence of neutrophil-specific annexin protein (39-kDa protein), we found that these cells seemed to be very similar to human peripheral polymorphonuclear leukocytes (PPMN), although they were in rather young stages. Stimulation-coupled responses of these cells were observed in terms of superoxide (O2.-) genration, luminol chemiluminescence response (LCL), membrane depolarization, and changes in intracellular calcium ion concentration ([Ca2+]i). The rates of superoxide generation by various stimuli, such as formylmethionylleucylphenylalanine (FMLP), phorbol 12-myristate 13-acetate (PMA) and opsonized zymosan (OZ) were different. Superoxide generation and strong chemiluminescence response were observed without addition of any stimuli. This endogenous LCL was inhibited by azide and superoxide dismutase (SOD), but not by uric acid (UA). The intensity of the endogenous LCL decreased with time after isolation from the oral cavity. This decrease was accompanied by the appearance of a FMLP-coupled response. Furthermore, the endogenous activity which produced active oxygen species was maintained in the medium at 4 degrees C for a long period after isolation. From these results, it is suggested that SPMN have the ability to show characteristic responses to various stimuli, and that SPMN play important roles in the defense mechanisms in the oral cavity.     

Characterization of the priming effects of human granulocyte-macrophage colony-stimulating factor on human neutrophil leukotriene synthesis

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an in vitro and in vivo stimulator of human bone marrow myelomonocytic precursor cells and mature granulocyte and macrophage effector cells. We have compared the effect of GM-CSF on the synthesis of 5-lipoxygenase products induced by the chemotactic peptide fMet-Leu-Phe and the calcium ionophore A23187 in human neutrophils. Although GM-CSF alone did not stimulate detectable synthesis of products of the 5-lipoxygenase pathway, pre-incubation of neutrophils with 200 pM GM-CSF for 1 hour at 23 degrees C enhanced synthesis of leukotriene B4, its all-trans isomers and omega-oxidation products, and 5-hydroxyeicosatetraenoic acid in response to both the calcium ionophore A23187 (1.5 microM), and the chemotactic peptide fMet-Leu-Phe (0.1 microM). This priming effect of GM-CSF was maximal after a 60 min incubation at 23 degrees C, or after a 30 min preincubation at 37 degrees C. The effect of GM-CSF was maximal using a concentration of 1 nM. Enhancement of the leukotriene synthesis stimulated by A23187 was only observed when the cells were stimulated by the ionophore for periods of 3 minutes or less. In contrast, the enhancing effect of GM-CSF was still apparent when cells were exposed to fMet-Leu-Phe for as long as 15 minutes. Furthermore, the enhancing effect of GM-CSF was ablated when neutrophils were stimulated with A23187 and exogenous arachidonic acid. However, co-addition of exogenous arachidonic acid with fMet-Leu-Phe did not entirely mask the effect of GM-CSF. Possible mechanisms of action of GM-CSF are discussed.     

Functional coupling of adenine nucleotide translocase and mitochondrial creatine kinase is enhanced after exercise training in lung transplant skeletal muscle

Mechanisms responsible for limitation of exercise capacity in lung transplant recipients (LR) and benefits gained by exercise training were studied. Mitochondrial respiration parameters, energy transfer, and cell structure were assessed in vastus lateralis biopsies using the permeabilized fiber technique with histochemical and morphometric measurements. Twelve male controls (C) and 12 LR performed exercise training over 12 wk. Before exercise training, there were strong correlations between exercise capacity (maximal O(2) consumption and endurance time at 70% maximal power output) and cellular events, as assessed by percentage of type I fibers and apparent K(m) for exogenous ADP. Anticalcineurins were not involved in LR exercise limitation, since there were no differences in maximal mitochondrial rate of respiration before exercise training and no abnormalities in respiratory chain complexes compared with C. Training resulted in a significant increase in physiological parameters both at the cellular (apparent K(m) for exogenous ADP and stimulating effect of creatine) and integrated (maximal O(2) consumption, power output at ventilatory threshold, maximal power output, and endurance time at 70% maximal power output) levels in LR and C. After the training period, improvements in maximal O(2) consumption and in maximal mitochondrial rate of respiration were noted, as well as changes in endurance time and percentage of type I fibers. Because there were no changes in diameters and fiber types, baseline alteration of apparent K(m) for exogenous ADP and its improvement after training might be related to changes within the intracellular energetic units. After the training period, intracellular energetic units exhibited a higher control of mitochondrial respiration by creatine linked to a more efficient functional coupling adenine nucleotide translocase-mitochondrial creatine kinase, resulting in better exercise performances in C and LR.     

Glycated Proteins Can Enhance Photooxidative Stress in Aged and Diabetic Lenses

This study intends to clarify the ability of different carbonyl-containing lens metabolites to form advanced glycation end products, which possess photosensitizer activity and to investigate whether these modified proteins could be implicated in lens photodamage. Calf lens protein was experimentally glycated with either methylglyoxal, glyoxal, ascorbic acid, or fructose to obtain models of aged and diabetic cataractous lenses. Being exposed to 200 J/cm 2 UVA radiation the model glycated proteins produced 2-3-fold more singlet oxygen compared to the unmodified protein and the superoxide radical formation was 30-80% higher than by the native protein. Ascorbylated proteins demonstrated the highest photosensitizer activity. Biological responses of glycation-related photosensitizers were studied on cultured lens epithelial cells irradiated with 40 J/cm 2 UVA. Tissue culture studies revealed a significant increase in thiobarbituric acid reactive substances in the culture medium of lens epithelial cells after irradiation and treatment with glycated proteins. Lens proteins had a protective effect against UVA induced cytotoxicity, however, this protective effect decreased with the increasing photosensitizer activity of experimentally glycated proteins. The documented glycation-related photosensitization could explain the accelerated pathogenic changes in human lens at advanced age and under diabetic conditions.     

Jasmonates modulate the promotion effects induced by SNP on root development of wheat under osmotic stress through lipoxygenase activation

We investigated promotion effects of exogenous sodium nitroprusside (SNP) on wheat seedling (Triticum aestivum L.) lateral root (LR) and root hair development, and the relationship between endogenous jasmonate (JA) production and activity changes of lipoxygenase (LOX) isoenzymes under osmotic stress generated by 15 % PEG-6000. Our results showed that 25 or 50 μM SNP could significantly increase LR length and number whether or not the seedlings were under PEG stress. When 50 μM cPTIO, 50 μM SHAM or 50 μM NDGA was supplemented, the promotion effects of SNP were blocked. SNP could also induce the production of endogenous JAs in roots, and 25 μM SNP induced the maximum JA content. The effect of SNP on JA production could also be blocked by adding cPTIO, SHAM or NDGA. Furthermore, the activity of lipoxygenase (LOX) in roots was affected by SNP; the maximal activity of LOX also occurred in the roots treated by 25 μM SNP under PEG stress, or 50 μM SNP without PEG stress. LOX isoenzymes in roots were detected by electrophoresis; the results showed that 25 μM SNP could noticeably increase the activities of LOXII and LOXIII under PEG stress. Our results suggest that, under osmotic stress generated by PEG, the promotion effects of exogenous SNP on wheat LR and root hair development could be mediated by endogenous JAs through LOX activation.     

A 41 kDa transferrin related molecule acts as an autocrine growth factor for HL-60 cells

HL-60 cells produce an autostimulatory growth factor. Since the stimulatory effect of HL-60 conditioned medium is only observed in the absence of exogenous transferrin we have assayed HL-60 cells for the production of transferrin and found that they produce polypeptides which react with transferrin antibodies. 35S-methionine labelling, immunoprecipitation and subsequent separation by SDS-gel electrophoresis reveals the presence of a major transferrin related 41 +/- 2 kDa species released by HL-60 cells. Physiological levels of iron salts completely abolish the requirement of exogenous transferrin which indicates that the endogenous transferrin related polypeptides in the presence of exogenous inorganic iron salts are sufficient for the proliferation of HL-60 cells provided insulin or related growth factors are present. The addition of transferrin receptor antibodies inhibits the stimulatory action of the endogenous transferrin related activity.     

CD4-CD8- human T cells: phenotypic heterogeneity and activation requirements of freshly isolated "double-negative" T cells

In the present study we have analyzed the in vitro activation requirements of freshly isolated CD4-CD8- "double-negative" (DN) human peripheral blood T cells. DN cells were isolated from E+ cells by removal of CD4+, CD8+, and CD16+ cells through consecutive steps of C'-mediated lysis and panning. While the majority (79.0 +/- 12.0%) of DN cells were TCR gamma delta+ as shown by staining with mAb TCR delta-1, a minor fraction (6.7 +/- 4.7%) expressed TCR alpha beta as revealed by staining with mAb BMA031. Within the gamma delta+ DN fraction, most cells reacted with mAb Ti gamma A which delineates a V gamma 9JPC gamma 1 epitope, whereas a minor fraction stained with mAb delta TCS-1 which identifies a V delta 1J delta 1 epitope. Functional studies performed at low cell number (1000) per microculture indicated that DN cells can be activated by anti-CD3 mAb, PHA and allogeneic stimulator cells, provided that exogenous growth factors are supplied. Both rIl-2 and rIl-4 acted as efficient growth factors for DN cells, and a synergistic stimulatory effect of rIl-2 and rIl-4 was observed when DN cells were cocultured with allogeneic LCL stimulator cells. As compared to unseparated E+ cells, isolated DN responder cells had a reduced capacity to secrete Il-2 upon PHA stimulation in the presence of LCL feeder cells. The majority of DN cells maintained their CD3+ CD4-CD8- phenotype upon coculture with allogeneic LCL stimulator cells. These data demonstrate that CD3+ DN cells in human peripheral blood are heterogeneous with respect to TCR expression. In addition, they show that freshly isolated DN cells are deficient in Il-2 production but may be normally stimulated by anti-CD3, PHA, or alloantigen if exogenous growth factors (rIL-2 and/or rIl-4) are provided.     

Neutrophil-derived oxidants as mediators of chemical activation in bone marrow

Neutrophil-derived oxidants have been implicated in both damage to biomolecules and the metabolic activation of xenobiotics. Since the bone marrow is a relatively neutrophil-rich tissue which is subject to xenobiotic toxicity, we have characterized the oxidant generating capability of neutrophilic cells isolated from femurs of male C57BL/6J mice. Addition of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to neutrophil preparations (70 +/- 5% ring neutrophils and metamyelocytes) elicited superoxide anion generation, as indicated by superoxide dismutase (SOD)-inhibitable acetylated cytochrome c reduction, and oxidant-dependent chemiluminescence (CL) from luminol or lucigenin. The interaction of benzo[a]pyrene-7,8-dihydrodiol (BP-diol), a proximate carcinogenic metabolite of benzo[a]pyrene (BP), with TPA-stimulated bone marrow neutrophils resulted in azide-inhibitable CL (90%) indicative of its myeloperoxidase-dependent oxidation to an excited-state intermediate. Covalent binding of [3H]BP-diol to exogenous DNA was similarly increased 3-fold in the presence of TPA-stimulated bone marrow neutrophils. Recently, our laboratory has shown that in addition to CL, TPA-stimulated human polymorphonuclear leukocytes can activate BP-diol to an intermediate which covalently binds to DNA and elicits mutagenicity in Salmonella typhimurium TA100. These observations combined with our current results suggest a possible role for neutrophil-derived oxidants in the mechanisms of chemically-induced bone marrow toxicity.     

Photosensitized inactivation of HeLa tumor cells by phthalocyanines

The combined effect of tetrasulfonated phthalocyanine (TSPC, 1.0 microM) or chloroaluminum phthalocyanine (CAPC, 1.0 microM) and copper vapour laser radiation (lambda = 670 nm) causes a dose dependent decrease in the survival rate of HeLa cells at exponential and stationary growth phases estimated by the trypan blue exclusion test or colony-forming ability test. TSPC is two times and CAPC seven times more effective, with regard to lethality, than the known photosensitizer, a hematoporphyrin derivative.     

Mechanisms of photochemotherapy-induced apoptotic cell death in lymphoid cells.

Previous studies we performed showed that 8-methoxypsoralen in combination with ultraviolet A light (photochemotherapy) caused DNA damage and that this caused nucleotide depletion in peripheral blood leukocytes, secondary to an active form of programmed cell death, poly(ADP-ribosyl)ation. Further studies revealed that 24 h after exposure to 10 J/cm2 ultraviolet A light and 8-methoxypsoralen (300 ng/mL), apoptotic cells increased from 3 (control) to 31% (p less than 0.001). Ultraviolet A light alone also significantly increased the number of apoptotic cells. These morphological changes were confirmed by parallel findings on DNA electrophoresis. Treatment with 2 to 5 J/cm2 of ultraviolet A light and 8-methoxypsoralen caused an approximately 30% increase in cytosolic free calcium levels in peripheral blood leukocytes 1 h after exposure. Associated with this was a 51% increase in 45Ca2+ uptake over the first 60 min. Similar findings in a different lymphoid cell (CCRF-CEM) confirmed the results obtained with peripheral blood leukocytes. The use of calcium-free medium prevented a rise in cytosolic free calcium and decreased the number of cells undergoing apoptotic cell death. Cycloheximide inhibited ultraviolet A light - 8-methoxypsoralen induced apoptosis in CCRF-CEM cells; it also decreased calcium levels in control CCRF-CEM cells. This study shows that ultraviolet A light - 8-methoxypsoralen caused apoptotic cell death in lymphoid cells; this appeared to be associated with calcium influx, presumably because of the requirement of endogenous endonucleases for calcium.