TWEAK/Fn14 Signaling Involvement in the Pathogenesis of Cutaneous Disease in the MRL/lpr Model of Spontaneous Lupus.

Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK, TNFSF12) and its sole receptor Fn14, belonging to the TNF ligand and receptor superfamilies respectively, are involved in cell survival and cytokine production. The role of TWEAK/Fn14 interactions in the pathogenesis of cutaneous lupus has not been explored. TWEAK treatment of murine PAM212 keratinocytes stimulated the secretion of RANTES via Fn14 and promoted apoptosis. Parthenolide, but not wortmanin or the MAPK inhibitor PD98059, significantly decreased production of RANTES, indicating that this effect of TWEAK is mediated via NF-κB signaling. UVB irradiation significantly upregulated the expression of Fn14 on keratinocytes in vitro and in vivo and increased RANTES production. MRL/lpr Fn14 knockout (KO) lupus mice were compared with MRL/lpr Fn14 wild-type (WT) mice to evaluate for any possible differences in the severity of cutaneous lesions and the presence of infiltrating immune cells. MRL/lpr Fn14 KO mice had markedly attenuated cutaneous disease as compared with their Fn14 WT littermates, as evidenced by the well-maintained architecture of the skin and significantly decreased skin infiltration of T cells and macrophages. Our data strongly implicate TWEAK/Fn14 signaling in the pathogenesis of the cutaneous manifestations in the MRL/lpr model of spontaneous lupus and suggest a possible target for therapeutic intervention.

Phenotypic and functional characterization of ultraviolet radiation-induced regulatory T cells.

Sensitization through UV-exposed skin induces regulatory T cells (Treg). In contrast to the classical CD4+CD25+ Treg that act contact dependent, UV-induced Treg (UV-Treg) suppress via IL-10, indicating a distinct subtype that requires further characterization. Depletion studies revealed that UV-Treg express the glucocorticoid-induced TNF family-related receptor (GITR) and the surface molecule neuropilin-1. The injection of T cells from UV-tolerized mice after depletion of UV-Treg into naive recipients enabled a contact hypersensitivity response, indicating that tolerization also induces T effector cells. Adoptive transfer experiments using IL-10-deficient mice indicated that the IL-10 required for suppression is derived from UV-Treg and not from host-derived cells. Activation of UV-Treg is Ag specific, however, once activated suppression is nonspecific (bystander suppression). Hence, speculations exist about the therapeutic potential of Treg generated in response to Ag that are not necessarily the precise Ag driving the pathogenic process. Thus, we studied the consequences of multiple injections of 2,4-dintrofluorobenzene (DNFB)-specific Treg into ears of naive mice followed by multiple DNFB challenges. DNFB-specific Treg were injected once weekly into the left ears of naive mice and DNFB challenge was performed always 24 h later. After three injections, a challenging dose of DNFB was applied on the right ear. This resulted in pronounced ear swelling, indicating that the subsequent boosting of DNFB-specific Treg had caused sensitization of the naive mice against DNFB. These data demonstrate that UV-Treg express GITR and neuropilin-1 and act via bystander suppression. However, constant boosting of Treg with Ag doses in the challenging range results in final sensitization that might limit their therapeutic potential.

The importance of abrogation of G2-phase arrest in combined effect of TRAIL and ionizing radiation.

BACKGROUND: In this work we studied the relationship between the enhanced expression of DR5 receptor and the effect of combination of TRAIL and ionizing radiation on cell cycle arrest and apoptosis induction in human leukemia cell line HL-60. MATERIAL AND METHODS: DR5, APO2.7 and cell cycle were analyzed by flow cytometry. Proteins Bid and Mcl-1 were analyzed by Western-blotting. For clonogenic survival, colony assay on methylcellulose was used. RESULTS: Ionizing radiation caused significantly enhanced positivity of DR5 receptors 24 h after irradiation with high doses (6 and 8 Gy). An increase of DR5 receptor positivity after a dose of 2 Gy was not statistically significant and application of TRAIL 48 h after irradiation did not increase the apoptosis induction. However, a decrease of radiation-induced G(2) phase arrest and an increase of apoptosis were observed when TRAIL was applied 16 h before irradiation with the dose of 2 Gy. Incubation with 6 microg/l TRAIL for 16 h reduced D(0) value from 2.9 Gy to 1.5 Gy. The induction of apoptosis by TRAIL was accompanied by Bid cleavage and a decrease of antiapoptotic Mcl-1 16 h after incubation with TRAIL. CONCLUSION: TRAIL in concentration of 6 microg/l applied 16 h before irradiation by the dose of 1.5 Gy caused the death of 63% of clonogenic tumor cells, similarly as the dose of 2.9 Gy alone, which is in good correlation with the enhanced apoptosis induction.

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.

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.

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.

Postgestational lymphotoxin/lymphotoxin beta receptor interactions are essential for the presence of intestinal B lymphocytes.

Lymphotoxin (LT), a cytokine belonging to the TNF family, has established roles in the formation of secondary lymphoid structures and in the compartmentalization of T and B lymphocyte areas of the spleen. In this study, we examine the role of LT in directing the composition of intestinal lymphocytes. We report that mice deficient in LT have a normal composition of intestinal lamina propria (LP) T lymphocytes, and an absence of intestinal LP B lymphocytes. We further refine this observation to demonstrate that the interaction of LT with the LTbetaR is essential for the presence LP B lymphocytes. The LT/LTbetaR-dependent events relevant for the presence of LP B lymphocytes occur after birth, do not require the presence of Peyer's patches, lymph nodes, or the spleen; and therefore, are distinct and independent from the previously identified roles of LT/LTbetaR. The LT-dependent signal relevant for the presence of LP B lymphocytes is optimally supplied by a LT-sufficient B lymphocyte, and requires a LTbetaR-sufficient radio-resistant, non-bone marrow-derived cell. Based upon the severity of the deficit of LP B lymphocytes we observed, these novel LT/LTbetaR-dependent events are of primary importance in directing the entry and residence of LP B lymphocytes.

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.

Radiation-induced release of transforming growth factor alpha activates the epidermal growth factor receptor and mitogen-activated protein kinase pathway in carcinoma cells, leading to increased proliferation and protection from radiation-induced cell death.

Exposure of A431 squamous and MDA-MB-231 mammary carcinoma cells to ionizing radiation has been associated with short transient increases in epidermal growth factor receptor (EGFR) tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and c-Jun NH(2)-terminal kinase (JNK) pathways. Irradiation (2 Gy) of A431 and MDA-MB-231 cells caused immediate primary activations (0-10 min) of the EGFR and the MAPK and JNK pathways, which were surprisingly followed by later prolonged secondary activations (90-240 min). Primary and secondary activation of the EGFR was abolished by molecular inhibition of EGFR function. The primary and secondary activation of the MAPK pathway was abolished by molecular inhibition of either EGFR or Ras function. In contrast, molecular inhibition of EGFR function abolished the secondary but not the primary activation of the JNK pathway. Inhibition of tumor necrosis factor alpha receptor function by use of neutralizing monoclonal antibodies blunted primary activation of the JNK pathway. Addition of a neutralizing monoclonal antibody versus transforming growth factor alpha (TGFalpha) had no effect on the primary activation of either the EGFR or the MAPK and JNK pathways after irradiation but abolished the secondary activation of EGFR, MAPK, and JNK. Irradiation of cells increased pro-TGFalpha cleavage 120-180 min after exposure. In agreement with radiation-induced release of a soluble factor, activation of the EGFR and the MAPK and JNK pathways could be induced in nonirradiated cells by the transfer of media from irradiated cells 120 min after irradiation. The ability of the transferred media to cause MAPK and JNK activation was blocked when media were incubated with a neutralizing antibody to TGFalpha. Thus radiation causes primary and secondary activation of the EGFR and the MAPK and JNK pathways in autocrine-regulated carcinoma cells. Secondary activation of the EGFR and the MAPK and JNK pathways is dependent on radiation-induced cleavage and autocrine action of TGFalpha. Neutralization of TGFalpha function by an anti-TGFalpha antibody or inhibition of MAPK function by MEK1/2 inhibitors (PD98059 and U0126) radiosensitized A431 and MDA-MB-231 cells after irradiation in apoptosis, 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and clonogenic assays. These data demonstrate that disruption of the TGFalpha-EGFR-MAPK signaling module represents a strategy to decrease carcinoma cell growth and survival after irradiation.

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.

[The Fas/Fas-ligand system: implications in the antitumor immune response and in the activity of cytotoxic agents]. / Le système Fas/Fas-ligand: implications dans la réponse immunitaire antitumorale et dans l'activité des agents cytotoxiques.

Interaction of Fas-ligand (Fas-L) with the extracytoplasmic domain of the Fas receptor can induce Fas trimerization and activation of the apoptotic cell death process. Several molecular pathways that lead to apoptosis and some of their regulatory mechanisms have been identified. Fas-related membrane receptors that contain a death domain in their intracytoplasmic domain have been identified. They constitute a death receptor family (DR1 to DR5) whose first member is the TNFR1 receptor for TNF alpha. The Fas/Fas-L system plays a role in the cytotoxic activity of immune cells and the regulation of immune response amplitude. This system could be involved in the immune response to tumor cells and the cytotoxic activity of drugs and radiations. The expression of Fas-L on the plasma membrane of numerous tumor cells allow them, in vitro, to kill Fas-expressing immune cells. This observations has suggested that tumor cells used Fas-L to induce a specific immune tolerance. However, in vivo, Fas-L expression rather induces tumor cell rejection. The quantity of Fas-L expressed on tumor cells could determine whether tumor cells are tolerated or rejected. Cytotoxic drugs and radiations modulate Fas and Fas-L expression on tumor cells. The role of Fas/Fas-L interactions in the cytotoxicity of these agents remains poorly defined. It has been clearly shown, however, that low doses of cytotoxic drugs increase Fas expression on tumor cells, thereby improving their elimination by immune cells. Drug-induced modulation of Fas expression could provide new therapeutic strategies combining chemotherapy with immunotherapy.

UVB-induced association of tumor necrosis factor (TNF) receptor 1/TNF receptor-associated factor-2 mediates activation of Rel proteins.

Exposure of mammalian skin to UV light results in induced gene transcription, playing a role in inflammation, immunosuppression, and tumor promotion. One important group of transcription factors induced by UV radiation is composed of members of the Rel/NF-kappa B family, which are known to play a major role in the transcriptional activation of many genes encoding inflammatory cytokines, adhesion molecules, and viral proteins. However, the upstream events in the transduction of the UVB signal to Rel protein activity are, as yet, unknown. Here, we provide biochemical evidence that exposure of keratinocytes to UVB causes rapid association of tumor necrosis factor (TNF) receptor 1 with its downstream partner TRAF-2. The functional relevance of this association is demonstrated by experiments showing that expression of a dominant negative TNF receptor 1 or TRAF-2 protein inhibits UVB-induced Rel-dependent transcription. Inclusion of a neutralizing antibody toward TNF alpha has no effect on UVB activation of a Rel-responsive reporter gene. Therefore, UVB-induced activation of Rel proteins via TNF receptor 1, independent of ligand activation, is a key component in the UV response in keratinocytes.