The mandatory role of IL-10-producing and OX40 ligand-expressing mature Langerhans cells in local UVB-induced immunosuppression.

The mechanism underlying the local UVB-induced immunosuppression is a central issue to be clarified in photoimmunology. There have been reported a considerable number of cells and factors that participate in the sensitization phase-dependent suppression, including Langerhans cells (LCs), regulatory T cells, IL-10, and TNF-alpha. The recent important finding that LC-depleted mice rather exhibit enhanced contact hypersensitivity responses urged us to re-evaluate the role of LCs along with dermal dendritic cells (dDCs) in the mechanism of UVB-induced immunosuppression. We studied the surface expression of OX40 ligand (OX40L) and the intracellular expression of IL-10 in LCs and dDCs from UVB-irradiated (300 mJ/cm(2)) skin of BALB/c mice and those migrating to the regional lymph nodes from UVB-irradiated, hapten-painted mice. In epidermal and dermal cell suspensions prepared from the UVB-irradiated skin, LCs expressed OX40L as well as CD86 and produced IL-10 at a higher level than Langerin(-) dDCs. The UVB-induced immunosuppression was attenuated by the administration of IL-10-neutralizing or OX40L-blocking Abs. In mice whose UVB-irradiated, hapten-painted skin was dissected 1 d after hapten application, the contact hypersensitivity response was restored, because this treatment allowed dDCs but not LCs to migrate to the draining lymph nodes. Moreover, LC-depleted mice by using Langerin-diphtheria toxin receptor-knocked-in mice showed impaired UVB-induced immunosuppression. These results suggest that IL-10-producing and OX40L-expressing LCs in the UVB-exposed skin are mandatory for the induction of Ag-specific regulatory T cells.

The Glycoprotein M6a Is Associated with Invasiveness and Radioresistance of Glioblastoma Stem Cells.

Systematic recurrence of glioblastoma (GB) despite surgery and chemo-radiotherapy is due to GB stem cells (GBSC), which are particularly invasive and radioresistant. Therefore, there is a need to identify new factors that might be targeted to decrease GBSC invasive capabilities as well as radioresistance. Patient-derived GBSC were used in this study to demonstrate a higher expression of the glycoprotein M6a (GPM6A) in invasive GBSC compared to non-invasive cells. In 3D invasion assays performed on primary neurospheres of GBSC, we showed that blocking GPM6A expression by siRNA significantly reduced cell invasion. We also demonstrated a high correlation of GPM6A with the oncogenic protein tyrosine phosphatase, PTPRZ1, which regulates GPM6A expression and cell invasion. The results of our study also show that GPM6A and PTPRZ1 are crucial for GBSC sphere formation. Finally, we demonstrated that targeting GPM6A or PTPRZ1 in GBSC increases the radiosensitivity of GBSC. Our results suggest that blocking GPM6A or PTPRZ1 could represent an interesting approach in the treatment of glioblastoma since it would simultaneously target proliferation, invasion, and radioresistance.

Evidence that singlet oxygen-induced human T helper cell apoptosis is the basic mechanism of ultraviolet-A radiation phototherapy.

Ultraviolet A (UVA) irradiation is effectively used to treat patients with atopic dermatitis and other T cell mediated, inflammatory skin diseases. In the present study, successful phototherapy of atopic dermatitis was found to result from UVA radiation-induced apoptosis in skin-infiltrating T helper cells, leading to T cell depletion from eczematous skin. In vitro, UVA radiation-induced human T helper cell apoptosis was mediated through the FAS/FAS-ligand system, which was activated in irradiated T cells as a consequence of singlet oxygen generation. These studies demonstrate that singlet oxygen is a potent trigger for the induction of human T cell apoptosis. They also identify singlet oxygen generation as a fundamental mechanism of action operative in phototherapy.

Gpnmb is a melanosome-associated glycoprotein that contributes to melanocyte/keratinocyte adhesion in a RGD-dependent fashion.

Gpnmb is a glycosylated transmembrane protein implicated in the development of glaucoma in mice and melanoma in humans. It shares significant amino acid sequence homology with the melanosome protein Pmel-17. Its extracellular domain contains a RGD motif for binding to integrin and its intracellular domain has a putative endosomal and/or melanosomal-sorting motif. These features led us to posit that Gpnmb is associated with melanosomes and involved in cell adhesion. We showed that human Gpnmb is expressed constitutively by melanoma cell lines, primary-cultured melanocytes and epidermal melanocytes in situ, with most of it found intracellularly within melanosomes and to a lesser degree in lysosomes. Our newly developed monoclonal antibody revealed surface expression of Gpnmb on these pigment cells, albeit to a lesser degree than the intracellular fraction. Gpnmb expression was upregulated by UVA (but not UVB) irradiation and by alpha-melanocyte-stimulating hormone (MSH) (but not beta-MSH); its cell surface expression on melanocytes (but not on melanoma cells) was increased markedly by IFN-gamma and TNF-alpha. PAM212 keratinocytes adhered to immobilized Gpnmb in a RGD-dependent manner. These results indicate that Gpnmb is a melanosome-associated glycoprotein that contributes to the adhesion of melanocytes with keratinocytes.

Radiation-induced damage to mouse urothelial barrier.

PURPOSE: To determine changes of the urothelial barrier during the early as well as late radiation response in mouse urinary bladder. MATERIALS AND METHODS: Groups of mice were irradiated with a single dose of 20Gy and sacrificed between days 0 and 360. Urothelial cell numbers were counted, and the fraction of urothelium with a positive immunohistochemical signal for uroplakin-III (UP-III) on the luminal surface of the bladder was defined. Also, cytoplasmic UP-III staining signal in urothelium was quantified using an arbitrary score (0-3). RESULTS: Irradiation resulted in a significant decrease in the number of superficial umbrella cells during the early response phase (days 0-31) as well as during the initial late radiation reaction (days 90, 120). Progressive loss of the UP-III layer on the bladder luminal surface correlated with the decrease in the number of umbrella cells (p=0.002). Also, increased cytoplasmic staining of UP-III in the urothelium was seen after irradiation, correlating negatively with the reduction of the superficial UP-III layer (p<0.0001). CONCLUSION: Irradiation of the urinary bladder results in morphological impairment of the urothelial barrier that is associated with a loss of superficial umbrella cells during the early as well as initial late radiation response phase.

Overexpression of P-glycoprotein in mammalian tumor cell lines after fractionated X irradiation in vitro.

We observed that in vitro exposure of mammalian tumor cells to fractionated x irradiation results in the expression of drug resistance. The cause of this resistance was investigated in a series of Chinese hamster ovary cell lines that had survived exposure to multiple lethal doses of radiation. These cell lines had increased levels of P-glycoprotein (Pgp), the multidrug-resistance-associated membrane glycoprotein. Consistent with the classic multidrug resistance phenotype, they exhibited cross-resistance to multiple drugs, as well as sensitivity to reversal of vincristine resistance by verapamil. However, the cell lines showed no change in their sensitivity to x rays. Pgp overexpression occurred in these cells, despite a lack of Pgp gene amplification or of significant alteration in Pgp messenger RNA levels. Although the cause of increased Pgp levels is not yet known, these data suggest a biological basis for the clinical problem of drug resistance that can occur in previously irradiated tumors.

Dimerization of the P-glycoprotein in membranes.

Plasma membranes from a CHO cell line, CHRC5, which exhibits multidrug resistance was studied using radiation inactivation analysis. The P-glycoprotein content of the membrane was determined by Western blots. Irradiation resulted in the loss of P-glycoprotein. The dependence of this loss on radiation dose corresponded to a target size of 250 kDa which is the molecular mass of a dimer of the P-glycoprotein. This is strong evidence to indicate that the P-glycoprotein self associates in the membrane.

Vaccination of apoptotic Fas ligand-expressing tumors decreased antitumor responses by enhanced production of immunosuppressive cytokines.

Expression of Fas ligand (FasL) in tumors produced antitumor effects by generating both inflammation and T cell-mediated immunity, although the Fas/FasL interaction induces an apoptotic process of Fas-positive activated T cells. Our previous study, however, showed that immunization of mice with ultraviolet (UV)-treated FasL-expressing tumors rather induced immune suppression to the tumors, whereas mice rejected UV-untreated FasL-expressing tumors and developed protective immunity subsequently. Since dendritic cells (DCs) control tumor-specific immune responses in vivo, we examined a possible role of DCs in the immune suppression induced. Administration of DCs that were co-cultured with UV-treated FasL-expressing tumors did not influence the growth of parent tumors that were subsequently inoculated. Migration of immunocompetent cells into UV-treated FasL-expressing tumors was not significantly different from that into UV-untreated FasL-expressing tumors. However, production of immunosuppressive but not T helper type 1 cytokines was enhanced when UV-treated FasL-expressing tumors were administered. These data collectively suggest that the immune suppression induced by UV-treated FasL-expressing tumors was not attributable to tolerance of DCs, but due to cytokine-induced suppression of cell-mediated immunity.

Death receptor ligands: new strategies for combined treatment with ionizing radiation.

The major goal of modern radiation oncology is the achievement of a maximal tumor control with minimal normal tissue damage. However, normal tissue tolerance may preclude the application of tumoricidal radiation doses. In order to overcome this limitation, strategies either to increase normal tissue tolerance or to reduce the radiation dose required may prove beneficial. In this regard, attempts to minimize the required radiation dose by reducing the number of malignant clonogenic cells are promising. Therefore, therapies which induce programmed cell death (apoptosis) in tumor cells, may prove to be suitable approaches. TRAIL (TNFalpha-related apoptosis inducing ligand)/Apo2L is a very promising member of the family of death ligands. The ligand preferentially induces apoptotic cell death in a wide range of tumor cells but not in normal cells. TRAIL/Apo2L triggers apoptosis even in cells not undergoing apoptosis in response to radiation, since ionizing radiation induce apoptosis by a different pathway as death ligands although an overlapping set of molecules is involved. Combination of both modalities has been shown to induce additive or synergistic apoptotic effects and eradication of clonogenic tumor cells thereby increasing the therapeutic efficacy. The present article reviews this novel biological strategy for optimized radiotherapy based on the combination of ionizing irradiation and death receptor triggered cell death.

Ionizing radiation alters Fas antigen ligand at the cell surface and on exfoliated plasma membrane-derived vesicles: implications for apoptosis and intercellular signaling.

Resident proteins that reside on the plasma membrane are continually exfoliated from the cell surface. Exfoliation is a selective, energy-dependent process that mediates intercellular communication. Ionizing radiation modulates the expression of many plasma membrane-bound growth regulators, including the "death" ligand, TNFSF6 (formerly known as FasL, CD95L). Here we report that ionizing radiation induces dose-dependent up-regulation of TNFSF6 on plasma membranes purified from SW620 cells, a TNFSF6-expressing colon cancer cell line. Serum-free medium conditioned by exposed and control cells was collected and exfoliated vesicles were obtained by ultracentrifugation. Western blot analysis of vesicles from unexposed cells and from cells treated with 10 Gy showed increased amounts of TNFSF6 compared to that on vesicles from unexposed cells. Cells treated with 4 Gy released vesicles having a low level of TNFSF6 on their surface relative to that on vesicles exfoliated from unexposed cells. When assayed for bioactivity, vesicles from unexposed cells induced the greatest level of apoptosis in TNFRSF6 (formerly known as FAS) receptor-bearing Jurkat cells (cell surviving fraction of 43.7 +/- 6.1; P < 0.05), followed by vesicles collected from cells treated with 4 Gy (79.6 +/- 2.6%; P < 0.05). Despite having a high level of TNFSF6 by Western analysis, vesicles collected from cells exposed to 10 Gy display minimal biological activity (77.9 +/- 3.2%; P < 0.05), suggesting that modification of the vesicle-associated ligand has occurred. Our results indicate that ionizing radiation increases the level of TNFSF6 exfoliated on extracellular vesicles. The data may provide a mechanism for abscopal and bystander effects after irradiation.

Molecular control of melanogenesis in malignant melanoma: functional assessment of tyrosinase and lamp gene families by UV exposure and gene co-transfection, and cloning of a cDNA encoding calnexin, a possible melanogenesis "chaperone".

Melanogenesis is a cascade of events significantly controlled by regulatory genes which are associated with the melanosomal membrane. This report introduces our current research efforts dealing with (a) the gene and protein expressions of tyrosinase and Lamp (lysosome-associated membrane protein) families by human melanoma cells after repeated exposures to UV light, (b) the coordinated alterations in the expression of the Lamp family gene and its encoding product after transfection of two genes of the tyrosinase family in human melanoma cells and (c) cloning and sequencing of a Ca(2+)-binding phosphoprotein, calnexin, which could be a candidate as a chaperone for sorting and maturation of tyrosinase and Lamp family glycoproteins in melanogenesis cascade. Our UV exposure study, as well as gene transfection and antisense hybridization experiments, has clearly indicated a marked and coordinated interaction of the Lamp-1 gene with the tyrosinase and TRP-1 genes in this process. We propose that melanogenesis is controlled at least by two major gene family products, i.e., (a) the tyrosinase family of tyrosinase, TRP-1 and TRP-2, and the Lamp family of Lamp-1, Lamp-2 and Lamp-3. These two gene families probably derived from primordial melanogenesis-associated genes which are common or closely related to each other.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is expressed in normal skin and cutaneous inflammatory diseases, but not in chronically UV-exposed skin and non-melanoma skin cancer.

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) is a member of the tumor necrosis factor family that preferentially induces apoptosis in transformed but not normal cells and that is constitutively expressed in many organs including the skin. In addition to its therapeutic potential, TRAIL might act as a natural guardian eliminating transformed cells at an early stage. Ultraviolet (UV) radiation is not only a potent carcinogen because of its mutagenic effects but also because of its capacity to paralyze natural protection mechanisms, including the tumor suppressor gene p53. Therefore, we studied the effect of UV exposure on the expression of TRAIL in the skin by immunohistochemical analysis. TRAIL and its receptors TRAIL-R1 and TRAIL-R4 were constitutively expressed in normal epidermis and not altered in a variety of inflammatory dermatoses including those associated with interface dermatitis. TRAIL was not altered in biopsies of acute sunburn, polymorphic light eruption, and photoprovocation testing, indicating that acute UV exposure does not affect TRAIL expression. No differences were observed in UV-protected and chronically UV-exposed skin samples of younger adults. In contrast, TRAIL was significantly reduced in chronically UV-exposed skin of elderly individuals. In addition, TRAIL expression was reduced in actinic keratoses and Bowen disease and almost completely lost in basal cell and squamous cell carcinomas. In contrast, keratoacanthomas did not reveal any alterations in TRAIL expression. Taken together, these data indicate that chronic UV exposure in elderly patients results in the loss of TRAIL expression, which might contribute to the increased risk of skin cancer in this population. Down-regulation of TRAIL might represent another example of a natural protection mechanism that is eliminated by chronic UV exposure.

In vitro effects of topotecan and ionizing radiation on TRAIL/Apo2L-mediated apoptosis in malignant glioma.

The survival of patients with malignant gliomas is still unsatisfactory despite multimodality treatment, therefore new therapeutic strategies are required. Tumor necrosis factor apoptosis related ligand (TRAIL/Apo2L), a member of the tumor necrosis factor superfamily, may induce apoptotic cell death in several tumors, but not in normal cells, upon binding with specific receptors. In the present study, the expression and function of TRAIL receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5) has been investigated in five human glioma cell lines (U87, U138, U373, A172, SW1783) in ex vivo tumors and in primary cultures obtained from the tumors. Our data show that gliomas preferentially express TRAIL R2 and that treatment with topotecan, a topoisomerase I inhibitor, significantly up-regulates its expression as detected by flow cytometry and western blotting. Moreover, in most cases, treatment with topotecan resulted in an increased sensitivity to TRAIL-dependent apoptosis, although cyclohexymide had to be added to induce apoptosis. On glioma cell lines, the effects of irradiation on TRAIL receptors were also analysed. In our experimental conditions, irradiation with 2 Gy had a modest additive effect on TRAIL-dependent apoptosis and was not able to modulate TRAIL receptor expression.

A cooperative model for Ca(++) efflux windowing from cell membranes exposed to electromagnetic radiation.

We propose a simplified version of a cooperative lattice membrane model given by Grodsky to explain observed Ca(++) efflux windowing effects from cell membranes exposed to electromagnetic radiation. Assuming that field induced conformational interactions occur only between bistable receptor sites and glycoprotein Ca(++) sites on the surface of the membrane, the model is shown to be equivalent to an Ising model. This model is known to have a phase transition to an ordered state in which a macroscopic number of Ca(++) sites are either occupied or unoccupied. We identify such states with enhanced Ca(++) efflux from cell membranes. By further assuming an averaged signal, sinusoidally varying coupling between receptor and Ca(++) sites and a power-law dependence of the characteristic time constant on the induced power-density of the applied field, we show that the model is consistent with published experimental results on power density windowing effects for particular values of model parameters. For these parameter values, the model predicts further power densities where windowing effects may be observed under appropriate conditions.

Radiation inactivation analysis of fusion and hemolysis by vesicular stomatitis virus.

Radiation inactivation analysis was used to determine the size of the functional unit responsible for fusion of vesicular stomatitis virus (VSV) with cardiolipin or phosphatidylcholine-phosphatidylethanolamine (1:1) liposomes, and for VSV-induced hemolysis. When radiation-insensitive background values were subtracted, the calculated functional units for all three activities were similar, ranging from 866 to 957 kDa, equivalent to about 15 G protein molecules. This is in striking contrast to results of similar studies with influenza and Sendai viruses, in which the functional unit corresponded in size to a single fusion protein monomer, and suggests that VSV fusion may occur by a different mechanism.

Effects of irradiation and tunicamycin on the surface glycoproteins of Schistosoma mansoni.

The cercarial glycocalyx and schistosomulum surface contains a number of glycoproteins which are expressed in very variable amounts within a parasite population. Tunicamycin inhibits glycoprotein synthesis of schistosomula if the parasites are incubated for 24 hr with the drug (10 micrograms ml-1). An unexpected increase in lectin binding to the parasite surface was observed but no other changes were detected. Schistosomula treated in this way did not develop in the host past the lung stage. Ultraviolet irradiation (400 microW min cm-2) also inhibited glycoprotein synthesis. Synthesis of other proteins, and in particular heat shock proteins, were also inhibited. Sera from mice (NIH strain) infected with irradiated cercariae contained antibodies which bound to normal schistosomula with lower affinity than to irradiated parasites. This is evidence that irradiation modifies the surface and secreted glycoproteins of schistosomula, so they are processed in a different way to normal glycoproteins by the host's immune system. The effects of irradiation on heat shock protein synthesis may allow the parasite to release a variety of proteins and glycoproteins in abnormal conformations. This may explain the enhanced immunogenicity of irradiated cercariae.

The infectivity of spongiform encephalopathies: does a modified membrane hypothesis account for lack of immune response?

Scrapie, the prototype of a group of diseases which have the unique property of being both hereditary and infectious, is also exceptional in that it fails to evoke an immune response. Purification of crude scrapie preparations revealed a strong association of infectivity with a membrane protein ('PrPsc'); but a protein with the same amino acid sequence ('PrPc') was subsequently also found in normal mammalian nervous tissue. It is postulated by some investigators that 'PrPsc' is itself the infectious agent, or the most important part thereof, but in papers making that proposal immunological aspects have not been addressed. Experimental evidence supporting the hypothesis of a membrane fragment as agent has likewise lately not been taken into account. A modified form of the membrane hypothesis could account for immunological as well as genetic aspects of these diseases.

Search for genes responsible for UV susceptibility of human cells: involvement of syndecan-1 in UV resistance.

UVr-1 UV-resistant cells were established from UV-sensitive human RSa cells. We looked for genes expressed differentially between UVr-1 and RSa cells using PCR-based mRNA differential display to elucidate the molecular mechanisms underlying UV resistance. The transcription levels of syndecan-1 mRNA were increased in UVr-1 cells compared with those of RSa cells. Syndecan-1 is a transmembrane heparan sulfate proteoglycan and associates with cell adhesion and the cytoskeleton. Flow cytometric analysis using anti-syndecan-1 monoclonal antibody revealed that syndecan-1 was more abundant in UVr-1 cells than in RSa cells. The MTT method revealed that UVr-1 cells treated with the antibody showed higher sensitivity to UV cell killing than mock-treated cells. Studies using antisense oligonucleotides for syndecan-1 showed that antisense-treated UVr-1 cells became sensitive to UV cell killing. Thus, syndecan-1 might be involved in UV resistance in UVr-1 cells.

Adenovirus-mediated transfer of Fas ligand gene augments radiation-induced apoptosis in U-373MG glioma cells.

Most malignant astrocytomas (gliomas) express a high level of Fas, whereas the surrounding normal tissues such as neurons and astrocytes express a very low level of Fas. Thus, transduction of Fas ligand would selectively kill malignant astrocytoma cells. On the other hand, glioma cells harboring p53 mutation have been reported to be resistant to conventional therapies including radiation. To override the resistance mechanism of glioma cells with p53 mutation to radiation, we transduced U-373MG malignant astrocytoma (glioma) cells harboring mutant p53 with Fas ligand via an adenovirus (Adv) vector in combination with X-ray irradiation, and evaluated the degree of apoptosis. The degree of apoptosis in U-373MG cells infected with the Adv for Fas ligand (Adv-FL) and treated with irradiation (81%) was much higher than that in U-373MG cells infected with Adv-FL and not treated with irradiation (0.8%) or that in U-373MG cells infected with the control Adv for lacZ and treated with irradiation (5.0%). In U-373MG cells infected with Adv-FL, irradiation increased the expression of Fas ligand. Coincident with the increase in Fas ligand, there was a marked reduction in the caspase-3 level and a marked increase in the cleaved form of poly(ADP-ribose) polymerase (PARP), which are downstream components of Fas ligand-mediated apoptosis. This suggests that the enhanced activation of caspase-3 by the transduction of Fas ligand combined with irradiation, induced extensive apoptosis in U-373MG cells. In summary, transduction of Fas ligand may override the resistance mechanism to radiotherapy in glioma cells harboring p53 mutation.

Up-regulation of human epidermal Langerhans' cell B7-1 and B7-2 co-stimulatory molecules in vivo by solar-simulating irradiation.

Ultraviolet (UV) radiation impairs cutaneous immune functions and induces antigen-specific tolerance both locally at the irradiated skin site, as well as at distant skin sites and systemically. It has been postulated that in the local model, altered Langerhans' cells (LC) provide tolerogenic signals, and studies in vitro have indicated that UV radiation may down-regulate the expression of co-stimulatory molecules on the surface of these cells. To examine the effect of UV radiation on LC co-stimulatory molecules in vivo, we irradiated human volunteers with erythematogenic doses of solar-simulating UV radiation (SSR), and analyzed the expression of cell surface markers in dermatome skin samples obtained 1-72 h post-irradiation. For flow cytometric analysis, epidermal cell (EC) suspensions were prepared and double labeled with monoclonal antibodies against CD1a or HLA-DR, and B7-1 (CD80), B7-2 (CD86), ICAM-1 (CD54), ICAM-3 (CD50), LFA-3 (CD58), E-cadherin, or integrin-beta4 (CD104). In unirradiated control skin samples, keratinocytes (KC) expressed high levels of E-cadherin. LC expressed high levels of both E-cadherin and ICAM-3, and low levels of B7-2, LFA-3, ICAM-1, and integrin-beta4. Following SSR, a triphasic reaction pattern was seen: an immediate, down-regulatory phase prevailing 2-6 h post-irradiation, when the number of DR+ and CD1a+ cells were temporarily reduced; a delayed, up-regulatory phase in which the number of LC was increased and the expression intensities of CD1a, HLA-DR, B7-1, and B7-2 were strongly up-regulated, maximally evident 12-24 h after irradiation, but no more seen at 48 h; and a late phase at 72 h, in which an influx of monocytes and a concomitant rise in DR+ cells was recorded. We conclude that to understand real-life cutaneous UV immunology, studies in vitro need to be complemented with studies in vivo. In the case of LC, the effects of erythematogenic UV radiation in vivo on human LC B7 co-stimulatory molecules include an up-regulatory stage.