Expression Level of Membrane Markers CD5, CD19, and CD20 in B Cells after UV-Irradiation and Incubation in the Presence of Autologous Plasma.

The effect of UV-light (240-390 nm) in doses of 151 and 755 J/m2 on the expression of membrane markers CD5, CD19, CD20 in human peripheral blood B cells was studied by flow cytometry. In 24 h after exposure to UV light, we observed activation of processes accompanied by structural rearrangements of B-cell membranes leading to changes in the expression of receptor molecules: the content of of CD19 and CD20 increased due to activation of the synthesis of these proteins, while the content of CD5 decreased. The percentage of CD5+ cells decreased over 24 h after UV-irradiation of lymphocytes, while addition of autologous plasma to the incubation medium produced a photoprotective effect on CD5+ cells.

Photocleavable Cadherin Inhibits Cell-to-Cell Mechanotransduction by Light.

Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved via mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.

[The Influence of UV-Light on the Sub-Populational Composition and Expression of Membrane Markers of Lymphocytes of Donor Blood].

The influence of UV-light (240-390 nm) at dozes of 151 and 755 J/m2 on the content of membrane markers of lymphocytes using the method of flow cytometry was investigated. It was demonstrated that during incubation of UV-irradiated lymphocytes the change of their populational and sub-populational composition occurs. Expression of complexes of CD3, CD 19,.CD8, CD 16, CD25 and CD95 increased. This increase was caused mainly by de novo synthesis. UV-light had immunostimulating effect on CD8+ T-lymphocyte population. Together with the increase of cytotoxic cells and NK-cells, activation of lymphocytes (increased amount of CD25+ and CD95+ cells) took place. Amount of cells undergone apoptosis or necrosis increased proportionally to the dosage. These changes were more expressed during incubation of lymphocytes in nutrition medium without autological blood serum, e.g. under deficiency of growth factors and antioxidants.

Evaluation of the photooxidation of galactosyl- and lactosylceramide by electrospray ionization mass spectrometry.

RATIONALE: Glycosphingolipids are important lipid molecules namely as constituents of the plasma membrane organized in lipid rafts, in signal transduction, and cell-cell communication. Although many human diseases are associated with oxidative stress and lipid oxidation, a link between oxidative stress and modification of glycosphingolipids has never been addressed. METHODS: In this study, the structural changes caused by UVA-induced photooxidation of galactosyl- (GalCer) and lactosylceramide (LacCer) molecular species were studied by electrospray ionization mass spectrometry (ESI-MS and MS/MS), using a quadrupole time-of-flight (QTOF) mass spectrometer and high-performance liquid chromatography/tandem mass spectrometry with a C5 stationary phase (C5 HPLC/MS/MS) using a linear ion trap. RESULTS: ESI-MS spectra of GalCer and LacCer after photooxidation showed new ions with a mass shift of +32 Da when compared with the ions of the non-modified glycosphingolipids. These new species were assigned as hydroperoxyl derivatives, confirmed by HPLC/MS/MS and through FOX 2 assay. In the ESI-MS and LC/MS of lactosylceramide a new ion with lower m/z value, assigned as glucosylceramide (GlcCer) + 32 Da, was also detected and proposed to be formed due to oxidative cleavage of lactosyl moieties. ESI-MS/MS of the oxidized species allowed us to infer the presence of isomeric hydroperoxyl derivatives, with the hydroperoxyl moiety either linked to the sphingosine backbone or in the unsaturated acyl chain. Oxidation in the sugar moieties was observed in the case of LacCer, suggesting an oxidation via radical reactive oxygen species that can induce the oxidative cleavage of the lactosyl moiety. CONCLUSIONS: This study shows that glycosphingolipids are prone to oxidation and the identified mass spectrometry fingerprint of oxidized galactosyl- and lactosylceramide species will support their future identification in lipidomic studies of biological samples under oxidative conditions.

[Influence of chronic irradiation on the distribution of blood lymphocyte subpopulations among professionals of the atomic industry].

The aim of the study was to investigate the effects of chronic exposure to ionizing radiation on the cellular immunity of employees of the nuclear industry. Peripheral blood samples were studied in 195 employees of Physics and Power Engineering Institute (PPEI, Obninsk), who professionallycontacted with sources ofionizing radiation and were under individual dosimetric control. The median cumulative dose was 61.2 mSv, the average duration of work at the enterprise -27 ± 5 years. The control group consisted of 57 healthy individuals of a similar age and sex who did not have contact with sources of radiation. Indicators of the cellular immunity were determined by flow cytometry. Comparison of a cell-mediated immunity was conducted separately in the two age groups (20-40 and 41-70 years). The significant reduction inthe relative content of CD4+CD8 T-helper cells and the increase in the relative content of CD3-CD16, CD56+ NK-cells were found in both age groups of the PPEI employees in comparison with the age-matched control groups (p < 0.05). Separate analysis of the results in the low dose group (up to 50 mSv) demonstrated reducing the relative content of T-helper cells and increasing the proportion of NK-cells (as in the analysis of whole groups without taking into account the cumulative dose), as well as reducing the proportion of CD8+CD25+ activated lymphocytes in PPEI employees as compared to the age-matched control. Multiple regression analysis of the immunological parameters dependence on age and dose established a significant correlation of the relative content of CD3-CD19+ B-cells (r = -0.284, p = 2.9 x 10(-4)) and CD19+CD5+ B1-lymphocytes (r = -0.241, p = 0.002) with the dose of employees regardless of age, indicating the relationship of the changes in the B-cell component of immune system with the radiation factor.

[Influence of UV-light on the profile of receptors of lymphocytes of donated blood].

The influence of UV-light (240-390 nm) at the dozes of 151 and 755 J/m2 on the maintenance of membrane markers of lymphocytes: CD3, CD19, CD4, CD8, CD25, CD95 using the method of flow cytometry was investigated. It was demonstrated that UV-irradiation of lymphocytes induced the decrease in the number of CD3, CD4 and CD19 markers together with a simultaneous increase of the amount of CD8, CD25 receptors during the daily incubation of cells in a nutrient medium without plasma. When autologous plasma is used during incubation of both native and UV-irradiated lymphocytes, the percentage of cells with the specified markers is not different from the corresponding values in the cells immediately after their purification. This finding points at the important role of antioxidants of the blood plasma and growth factors for the maintenance of the sub-population structure of lymphocytes. However, the level of expression of CD95, one of the markers of apoptosis, increases in the photomodified cells incubated both in the presence and in the absence of plasma. This increase was revealed to be caused mainly by the synthesis of the protein de novo under the influence of apoptosis-inducing factors.

8-methoxypsoralen plus ultraviolet A therapy acts via inhibition of the IL-23/Th17 axis and induction of Foxp3+ regulatory T cells involving CTLA4 signaling in a psoriasis-like skin disorder.

To elucidate the molecular action of 8-methoxypsoralen plus UVA (PUVA), a standard dermatological therapy, we used K5.hTGF-beta1 transgenic mice exhibiting a skin phenotype and cytokine abnormalities with strong similarities to human psoriasis. We observed that impaired function of CD4+CD25+ regulatory T cells (Tregs) and increased cytokine levels of the IL-23/Th17 pathway were responsible for the psoriatic phenotype in this mouse model. Treatment of K5.hTGF-beta1 transgenic mice with PUVA suppressed the IL-23/Th17 pathway, Th1 milieu, as well as transcription factors STAT3 and orphan nuclear receptor RORgammat. PUVA induced the Th2 pathway and IL-10-producing CD4+CD25+Foxp3+Tregs with disease-suppressive activity that was abolished by anti-CTLA4 mAb treatment. These findings were paralleled by macroscopic and microscopic clearance of the diseased murine skin. Anti-IL-17 mAb treatment also diminished the psoriatic phenotype of the mice. This indicated that both induced Tregs involving CTLA4 signaling and inhibition of the IL-23/Th17 axis are central for the therapeutic action of PUVA.

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.

Effects of a new pathogen-reduction technology (Mirasol PRT) on functional aspects of platelet concentrates.

BACKGROUND: Several strategies are being developed to reduce the risk of pathogen transmission associated with platelet (PLT) transfusion. STUDY DESIGN AND METHODS: The impact of a new technology for pathogen reduction based on riboflavin plus illumination (Mirasol PRT, Navigant Biotechnologies, Inc.) at 6.2 and 12.3 J per mL on functional and biochemical characteristics of PLTs was evaluated. PLT concentrates (PCs) obtained by apheresis were treated with Mirasol PRT and stored at 22 degrees C. Modifications in major PLT glycoproteins (GPIbalpha, GPIV, and GPIIb-IIIa), adhesive ligands (von Willebrand factor [VWF], fibrinogen [Fg], and fibronectin), activation antigens (P-selectin and LIMP), and apoptotic markers (annexin V binding and factor [F]Va) were analyzed by flow cytometry. Adhesive and cohesive PLT functions were evaluated with well-established perfusion models. Studies were performed on the preparation day (Day 0) and during PCs storage (Days 3 and 5). RESULTS: Levels of glycoproteins remained stable during storage in PCs treated with 6.2 J per mL pathogen reduction technology (PRT) and similar to those observed in nontreated PCs. When 12.3 J per mL PRT was applied, however, levels of GPIbalpha moderately decreased on Days 3 and 5. VWF, Fg, and FVa were not modified in their expression levels, either by treatment or by storage period. Fibronectin appeared more elevated in all PRT samples. A progressive increase in P-selectin and LIMP expression and in annexin V binding was observed during storage of PRT-treated PCs. Functional studies indicated that 6.2 J per mL Mirasol PRT-treated PLTs preserved adhesive and cohesive functions to levels compatible with those observed in the respective control PCs. CONCLUSION: PLT function was well preserved in PCs treated with 6.2 J per mL Mirasol PRT and stored for 5 days.

Differentiation of monocytic U937 cells under static magnetic field exposure.

We present here a morphological, cytochemical and biochemical study of the macrophagic differentiation of human pro-monocytic U937 cells exposed to moderate intensity (6 mT) static magnetic fields (MF). It was found that the following substances induced differentiation in U937 cells to a progressively lower degree: 50 ng/mL 12-0-tetradecanoyl-13-phorbol acetate (TPA), low concentration of glutamine (0,05 mM/L), 10% dimethyl sulfoxide (DMSO) and 100 mM/L Zn++. Differentiated U937 cells shift from a round shape to a macrophage-like morphology, from suspension to adhesion growth and acquire phagocytotic activity, the cytoskeleton adapting accordingly. Exposure to static MF at 6 mT of intensity decreases the degree of differentiation for all differentiating molecules with a consequent fall in cell adhesion and increased polarization of pseudopodia and cytoplasmic protrusions. Differentiation alone, or in combination with exposure to static MFs, affects the distribution and quantity of cell surface sugar residues, the surface expression of markers of macrophage differentiation, and phagocytotic capability. Our results indicate that moderate-intensity static MFs exert a considerable effect on the process of macrophage differentiation of pro-monocytic U937 cells and suggest the need for further studies to investigate the in vivo possible harmful consequences of this.

Changed adhesion molecule profile of Ewing tumor cell lines and xenografts under the influence of ionizing radiation.

BACKGROUND: Adhesion molecules are involved in cell-cell and cell-matrix interactions and may be informative to characterize intercellular mechanisms of invasion and metastasis. This study was performed to characterize radiation-induced changes in the adhesion molecule profile of Ewing tumor subpopulations on a single cell level. MATERIALS AND METHODS: In the present study, two Ewing tumors were characterized in vitro 4, 24 and 72 hours after radiation with 5 Gy and in vivo in a xenograft model 4, 6 and 15 days after radiation with 30 Gy, together with non-irradiated controls, by five parameter flow cytometry. Directly fluorescence-conjugated antibodies that were directed against adhesion molecules (LFA-1 (CD11a), HCAM (CD44), VLA-2 (CD49b), ICAM-1 (CD54), NCAM (CD56), LECAM-1 (CD62L) and CD86) were used. Annexin V and 7-AAD were used to characterize radiation-induced apoptosis. RESULTS: Tumor cell subpopulations were identified by the expression of adhesion molecules, apoptotic markers and DNA content. Heterogeneous changes of the adhesion molecule profile were identified on tumor cell subpopulations after radiation. The expression of CD11a and CD62L correlated with the expression of apoptosis-associated markers. CONCLUSION: The changes of flow cytometric profile under radiation may potentially correlate with a changed metastatic potential of tumor cell subpopulations.

The effect of low dose gamma irradiation on the differentiation and maturation of monocyte derived dendritic cells.

The effects of gamma-irradiation on the differentiation of peripheral blood monocytes (PBM) into monocyte derived dendritic cells (MDC), their maturation, and subsequent ability to present antigen to T cells was studied. Undifferentiated MDC were more sensitive to gamma-irradiation induced apoptosis than mature MDC. Irradiation of immature MDC with 5 Gy of gamma-rays down regulated the expression of the costimulatory receptors CD80/CD86 and may compromise their ability to capture and present antigen. By contrast, gamma-Irradiation of mature MDC did not affect the expression of CD86/CD80, and HLA-DR. Gamma-irradiation increased the apoptosis of MDC; but did not affect the ability of mMDC to stimulate autologous MLR. T cell proliferative response in the MLR and in response to tetanus antigen was reduced when gamma-irradiated primary DC1 were used to either stimulate or present antigen to T cells.

Parenchymal, but not leukocyte, TNF receptor 2 mediates T cell-dependent hepatitis in mice.

TNF-alpha is a central mediator of T cell activation-induced hepatitis in mice, e.g., induced by Pseudomonas exotoxin A (PEA). In this in vivo mouse model of T cell-dependent hepatitis, liver injury depends on both TNFRs. Whereas TNFR1 can directly mediate hepatocyte death, the in vivo functions of TNFR2 in pathophysiology remained unclear. TNFR2 has been implicated in deleterious leukocyte activation in a transgenic mouse model and in enhancement of TNFR1-mediated cell death in cell lines. In this study, we clarify the role of hepatocyte- vs leukocyte-expressed TNFR2 in T cell-dependent liver injury in vivo, using the PEA-induced hepatitis model. Several types of TNFR2-expressing leukocytes, especially neutrophils and NK cells, accumulated within the liver throughout the pathogenic process. Surprisingly, only parenchymal TNFR2 expression, but not the TNFR2 expression on leukocytes, contributed to PEA-induced hepatitis, as shown by analysis of wild-type --> tnfr2 degrees and the reciprocal mouse bone marrow chimeras. Furthermore, PEA induced NF-kappaB activation and cytokine production in the livers of both wild-type and tnfr2 degrees mice, whereas only primary mouse hepatocytes from wild-type, but not from tnfr2 degrees, mice were susceptible to cell death induced by a combination of agonistic anti-TNFR1 and anti-TNFR2 Abs. Our results suggest that parenchymal, but not leukocyte, TNFR2 mediates T cell-dependent hepatitis in vivo. The activation of leukocytes does not appear to be disturbed by the absence of TNFR2.

Late effects of the Chernobyl radiation accident on T cell-mediated immunity in cleanup workers.

The main goal of this investigation was to evaluate the abnormal T-cell immunity in cleanup workers who took part in the cleanup after the Chernobyl accident in 1986. Peripheral blood mononuclear cells (MNCs) of apparently healthy cleanup workers (n = 134) were used to analyze the phenotype and proliferative response to mitogens in vitro. Evaluation of the MNC phenotype of cleanup workers did not reveal a significant disturbance in the T-cell subpopulation content except for an increase in CD3+CD16+56+ (NKT) cells. Immunophenotyping of phytohemagglutinin (PHA)-activated MNCs demonstrated suppression of CD4+ T-cell propagation and augmentation of CD8+ T-cell propagation in vitro compared to control individuals. DNA synthesis in the MNCs of cleanup workers was markedly inhibited after activation for 3 days with suboptimal concentrations of PHA, pokeweed mitogen and PMA. In contrast to control individuals, the monocytes of cleanup workers were able to stimulate the proliferation of T cells from healthy individuals but inhibited the proliferation of T cells from cleanup workers. This study affords a better understanding of the response of MNCs to stimulation with suboptimal concentrations of PHA and provides an approach to a more accurate analysis of the immunological disorders found after exposure to radiation from Chernobyl-related activities.

Uneven distributions of naïve and memory T cells in the CD4 and CD8 T-cell populations derived from a single stem cell in an atomic bomb survivor: implications for the origins of the memory T-cell pools in adulthood.

The processes that lead to the establishment and maintenance of memory T-cell pools in humans are not well understood. In this study, we examined the emergence of naïve and memory T cells in an adult male who was exposed to an atomic bomb radiation dose of approximately 2 Gy in 1945 at the age of 17. The analysis presented here was made possible by our earlier observation that this particular individual carries a hematopoietic stem cell mutation at the hypoxanthine phosphoribosyltransferase (HPRT) locus that is almost certainly a result of his exposure to A-bomb radiation. Our key finding is that we detected a very much higher HPRT mutant frequency in the naive (CD45RA(+)) cell component of this individual's CD4 and CD8 T-cell populations than in the memory (CD45RA(-)) cell component of his CD4 and CD8 T-cell populations. This stands in marked contrast to our finding that HPRT mutant frequencies are fairly similar in the naïve CD45RA(+) and memory CD45RA(-) components of the CD4 and CD8 T-cell populations of three unexposed individuals examined concurrently. In addition we found that the HPRT mutant frequencies were about 30-fold higher in the naïve (CD45RA(+)) CD4 T cells of the exposed individual than in his memory (CD45RA(-)) cell populations, but that the effect was a little less striking in his CD8 cell populations, where the HPRT mutant frequencies were only about 15-fold higher in his naïve T-cell pools than in his memory T-cell pools. We further found that 100% of the HPRT mutant cells in both his CD4 and CD8 naïve cell subsets appeared to have originated from repeated divisions of the initial HPRT mutant stem cell, whereas only 4 of 24 and 5 of 6 mutant cells in his CD4 and CD8 memory cell subsets appeared to have originated from that same stem cell. The most straightforward conclusion may be that the great majority of the T cells produced by this individual since he was 17 years old have remained as naïve-type T cells, rather than having become memory-type T cells. Thus the T cells that have been produced from the hematopoietic stem cells of this particular A-bomb-exposed individual seldom seem to enter and/or to remain in the memory T-cell pool for long periods. We speculate that this constraint on entry into memory T-cell pools may also apply to unirradiated individuals, but in the absence of genetic markers to assist us in obtaining evidential support, we must await clarifying information from radically different experimental approaches.

Effects of extremely low frequency (50 Hz) magnetic field on morphological and biochemical properties of human keratinocytes.

We investigated the effects on human keratinocytes (HaCaT) of exposure to a sinusoidal magnetic field of 2 mT (50 Hz). These cells are a good model for studying interaction of nonionising radiation, because they are not shielded from fields in vivo and also because they are resistant to both mechanical and thermal stimuli. We performed scanning microscopy which showed modification in shape and morphology in exposed cells. This modification is related to differential actin distribution as revealed by phalloidin fluorescence analysis. Moreover, the exposed cells show increased clonogenic capacity, as well as increased cellular growth as showed by clonogenicity assays and growth curves. Indirect immunofluorescence analysis using a fluorescent antibody against involucrin and beta4 integrin, which are respectively differentiation and adhesion markers, revealed an increase of involucrin expression and segregation of beta4 integrin in the cell membrane in cells exposed to 50 Hz; a higher percentage of the exposed cells shows a modified pattern of adhesion and differentiation markers. We also present evidence that exposure of HaCaT cells can interfere with protein kinase activity. Our observations confirm the hypothesis that electromagnetic fields at 50 Hz may modify cell membrane morphology and interfere with initiation of the signal cascade pathway and cellular adhesion.

Low-dose ultraviolet B radiation synergizes with TNF-alpha to induce apoptosis of keratinocytes.

High-dose ultraviolet B (UVB) irradiation is known to induce apoptosis of keratinocytes, but low-dose UVB dose not. In this paper we present evidence that low-dose UVB can induce TNF-alpha-dependent apoptosis of keratinocytes. In our study, 5 mJ/cm(2) doses of UVB were not sufficient by themselves to induce apoptosis of cultured human keratinocytes, but 20 mJ/cm(2) doses of UVB were. The combination of 5 mJ/cm(2) doses of UVB and exogenous TNF-alpha (15 ng/ml) induced significant apoptosis of keratinocytes, although exogenous TNF-alpha without UVB did not. This phenomenon was accompanied by enhanced clustering of tumor necrosis factor receptor 1 (TNFR1). TNF-alpha's promotion of the induction of apoptosis by low-dose UVB was seen until 30 min after irradiation but not at 1 h. We confirmed this finding using a skin organ culture system. UVB (20 mJ/cm(2)), which did not induce transformation of epidermal keratinocytes into sunburn cells, induced apoptosis when TNF-alpha was added to the culture medium. These results suggest that one of the possible mechanisms of inducing keratinocyte apoptosis by low-dose UVB and TNF-alpha is that low-dose UVB augments ligand-binding-induced TNFR1 clustering, resulting in increased apoptotic cell death.

Melphalan and other anticancer modalities up-regulate B7-1 gene expression in tumor cells.

In this study, we show that administration of low-dose melphalan (l -PAM, l -phenylalanine mustard) to mice bearing a large MOPC-315 plasmacytoma led to a rapid up-regulation of B7-1 (CD80), but not B7-2 (CD86), expression on the surface of MOPC-315 tumor cells. This l -PAM-induced preferential up-regulation of B7-1 surface expression was due, at least in part, to a direct effect of l -PAM on the tumor cells, as in vitro exposure of MOPC-315 tumor cells to l -PAM led to the preferential up-regulation of B7-1 surface expression. Moreover, in vitro exposure of MOPC-315 tumor cells to two other anticancer modalities, gamma-irradiation and mitomycin C, resulted in the preferential up-regulation of B7-1 surface expression. This effect was not restricted to MOPC-315 tumor cells, as preferential up-regulation of B7-1 surface expression was observed also following in vitro exposure of the P815 mastocytoma (that is negative for both B7-1 and B7-2 surface expression) to any of the three anticancer modalities. The up-regulation of B7-1 surface expression following in vitro exposure of tumor cells to l -PAM, gamma-irradiation, or mitomycin C required de novo protein and RNA synthesis, and was associated with the accumulation of mRNA for B7-1 within 4-8 h, indicating that the regulation of B7-1 expression is at the RNA transcriptional level. These results have important implications for an additional immune-potentiating mechanism of these anticancer modalities in clinical setting.

Ultraviolet-irradiation-induced apoptosis is mediated via ligand independent activation of tumor necrosis factor receptor 1.

Ultraviolet (UV)-irradiation has been shown to induce jun N-terminal kinase activity via aggregation-mediated activation of tumor necrosis factor receptor 1 (TNFR1) but the role of TNFR1 in mediating UV-induced apoptosis has not been explored. Using p53-null cells, we demonstrate that UV-stimulated ligand independent activation of TNFR1 plays a major role in mediating the apoptotic effects of UV-irradiation. UV-irradiation and TNF alpha acted in a synergistic manner to induce apoptosis. UV-irradiation stimulated the aggregation-mediated activation of TNFR1 which was coupled with activation of caspase 8, the most proximal caspase in TNF alpha signaling pathway. CrmA and the dominant negative versions of FADD, caspase 8 and caspase 10, that block the apoptotic axis of TNFR1 at different levels, also independently inhibited the UV-induced apoptosis. The engagement of the membrane initiated events was specific for UV-irradiation since neither CrmA nor the dominant negative FADD, caspase 8 or caspase 10 blocked the ionizing radiation-induced apoptosis. Cisplatin and melphalan, the UV-mimetic agents known to elicit UV-type DNA damage, also induced apoptosis but differed from UV in that both of the former agents engaged the caspase cascade at a level distal to FADD. Consistent with these findings cisplatin also did not stimulate TNFR1 aggregation. Together these results indicate that DNA damage per se was not sufficient to activate the membrane TNFR1. Based on our results we propose that the plasma membrane initiated events play a predominant role in mediating UV-irradiation-induced apoptosis and that UV-irradiation appears to engage the apoptotic axis of TNFR1 and perhaps those of other membrane death receptors to transduce its apoptotic signals.

Differential effect of phototherapy on the activities of human natural killer cells and cytotoxic T cells.

Exposure to ultraviolet B (UV-B) light is recognized to induce suppression of certain immune responses, particularly delayed hypersensitivity. However, its effect on cytotoxic T lymphocyte (CTL) activity, of major importance in the resistance to viruses and tumours, has not been assessed to the same extent. In this study five normal subjects, seropositive for herpes simplex virus (HSV), underwent a standard course of broadband UV-B therapy, as used in the treatment of psoriasis. They received whole-body irradiation thrice weekly for four weeks with incremental doses dependent on skin type. Blood samples were taken immediately before, at two time points during, and at the end of the therapy. An HSV-specific CTL assay was performed using autologous B cells transformed with Epstein-Barr virus as targets. No consistent modulation in CTL activity was obtained as a result of the therapy. The CTLs were separated into CD4 and CD8 subsets by positive selection and, again, no effect of irradiation on CTL activity within each of these two populations was observed. In contrast, the natural killer (NK) cell activity, assessed by the lysis of K562 cells, was significantly reduced at the first time point after the initiation of the phototherapy in all five subjects, and it continued to decline as the treatment progressed. Thus a differential effect of UV-B exposure on cytotoxic activity has been demonstrated: the HSV-specific CTL response is unchanged, while the NK response is suppressed.