Reduced IL-12 production by monocytes upon ultraviolet-B irradiation selectively limits activation of T helper-1 cells

The capacity of APC to stimulate the proliferation of human peripheral blood T cells decreases upon ultraviolet-B (UVB) irradiation. The aim of this study was to investigate whether all T cell subsets are equally sensitive to this reduced APC function. Established human Th1, Th2, and Th0 clones were stimulated with monocytes in a soluble CD3 mAb-mediated assay that is dependent on the presence of APC. Monocytes were exposed to low nonlethal doses of UVB radiation before coculture with T cells. UVB irradiation inhibited the capacity of monocytes to stimulate the proliferation and IFN-gamma production of Th1 cells in a dose-related fashion. In contrast, UVB-treated monocytes induced normal proliferation and IL-4 production in Th2 cells. Stimulation of Th0 cell proliferation by UVB-irradiated monocytes was normal, but a preferential suppression of IFN-gamma production was observed, thus leading to a more Th2-like cytokine response. The loss of Th1 proliferation upon stimulation with UVB-irradiated monocytes could be overcome by rIL-2; however, IFN-gamma production remained suppressed. IFN-gamma production could be completely restored by rIL-12, whereas the addition of IL-1 beta, TNF-alpha, or indomethacin had no such effect, nor did the addition of mAb to CD28, added to compensate for the reduced B7 expression of UVB-irradiated monocytes. Monocytes exposed to UVB radiation exhibited reduced expression of mRNA for the IL-1 2 subunits p35 and p40 and suppressed production of the IL-12 p70 protein. Our results thus indicate that UVB irradiation of APC selectively impairs Th1-like responses, a phenomenon caused by the UVB-induced suppression of monocyte IL-12 production.     

Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Calpha (PKCalpha), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCalpha. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4beta-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H2O2 levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.     

TNF receptor p55 plays a pivotal role in murine keratinocyte apoptosis induced by ultraviolet B irradiation

Excess exposure of skin to ultraviolet B (UVB) results in the appearance of so-called sunburn cells. Although it has been demonstrated that sunburn cells represent apoptotic keratinocytes, the molecular mechanisms for UVB-induced apoptosis in keratinocytes have not been fully elucidated. The cytokine, TNF-alpha, has been shown to induce apoptosis in a variety of cell types. Since UVB induces keratinocytes to release TNF-alpha, we hypothesized that TNF-alpha is involved in UVB-induced apoptosis in keratinocytes. In order to confirm this hypothesis and to further delineate which type of TNF receptor signaling mediates the apoptosis pathway, we performed both in vivo and in vitro experiments using gene-targeted knockout mice lacking either the TNF p55 receptor or the TNF p75 receptor. In the in vivo study, wild-type and mutant mice were exposed to UVB, and apoptotic keratinocytes were detected by examining DNA fragmentation using in situ nick-end labeling. For the in vitro experiments, keratinocytes derived from the wild-type and mutant mice were irradiated with UVB, and the degree of apoptosis was determined by flow cytometry, nick-end labeling of DNA, and a DNA ladder assay. Both in vivo and in vitro studies demonstrated that the deletion of TNF receptor p55 could suppress UVB-induced apoptosis in keratinocytes. Our observations support the notion that TNF-alpha is involved in UVB-induced keratinocyte apoptosis, and demonstrate that p55 receptor signaling plays a pivotal role in this event.     

Inhibition of ultraviolet B-induced activator protein-1 (AP-1) activity by aspirin in AP-1-luciferase transgenic mice

Aspirin is under consideration as a promising chemopreventative agent for human cancers. To study the usefulness of aspirin as a chemopreventative agent for UV-induced human skin cancer, we investigated the effect of aspirin on UVB-induced activator protein-1 (AP-1) activity. In the JB6 cell culture system, aspirin or sodium salicylate (SA) inhibited UVB-induced AP-1 activity in a dose-dependent manner; this inhibitory effect occurred only in cells pretreated with aspirin or SA before UVB irradiation but not cells treated with aspirin or SA after UVB irradiation. Furthermore, these inhibitory effects on UVB-induced AP-1 activity appeared to be mediated through blocking of activation of MAP kinase family members, including extracellular signal-regulated protein kinases, c-Jun N-terminal kinases, and p38. It was not due to absorption of UVB light by aspirin. In the skin of AP-1-luciferase transgenic mice, UVB irradiation induced a rapid increase in AP-1 activity, which reached the peak at 48 h post-UVB irradiation. The topical pretreatment of mouse skin with aspirin markedly blocked the UVB-induced AP-1 transactivation in vivo. These data provide the first evidence that aspirin and SA are inhibitors of UV-induced signal transduction and thus could be used as a chemopreventative agent for skin cancer.     

Determinants of asthma in a farming population

2-benzoyl-3-phenylquinoxaline 1,4-dioxide (BPQ) and other substituted quinoxaline 1,4-dioxides (QdO) were tested for their ability to inhibit the stimulations of ornithine decarboxylase (ODC) enzyme activity and DNA synthesis, two biochemical markers linked to skin tumour promotion by ultraviolet B (UVB) radiation. Topical application of BPQ on the dorsal skin of hairless mice was found to inhibit in a dose-dependent manner UVB-induced ODC activity and DNA synthesis. When applied 20 min before UVB radiation, a dose of 17 mg BPQ applied in 0.4 ml of vehicle inhibited UVB-induced ODC activity and DNA synthesis by 95% and 85%, respectively. This inhibitory effect is dependent on the time of administration of BPQ relative to UVB radiation, with a generally greater inhibition observed when this compound is applied before rather than after UVB treatment. The inhibitory abilities of the other QdO on the ODC and DNA responses induced by UVB radiation greatly varied and appear to be dependent on the structure of the compounds and their metabolic activation in the skin following irradiation. The remarkable effectiveness of BPQ against the ODC and DNA markers of UVB promotion is also observed following multiple applications of this agent. These results suggest that QdO, in particular BPQ and certain derivatives of it, may be useful in protecting the skin against UVB-induced skin damage.     

To see but not to read; the magnocellular theory of dyslexia

Ultraviolet (UV) radiation induces cytokine release from cultured keratinocytes as well as from epidermis in vivo. The purpose of this study was to determine whether differentiation of cultured keratinocytes into stratified epithelium decreases the effects of UVA and UVB radiation on cytokine release. Interleukin-1 (IL-1) alpha, IL-1 beta and tumor necrosis factor (TNF)-alpha release from human keratinocytes and reconstituted human epidermis was measured after exposure to UVA or UVB radiation. Release of IL-1 alpha, IL-1 beta, and TNF-alpha was induced by both UVA and UVB radiation from both keratinocytes and reconstituted epidermis. Release of these cytokines was correlated with cytotoxicity. Keratinocyte cultures were far more sensitive to UVB radiation than reconstituted epidermis, in terms of both cytotoxicity and cytokine release. In contrast, epidermal stratification/differentiation had much less effect on the sensitivity to UVA radiation. We conclude that epidermal stratification and the formation of a stratum corneum provide protection against UVB radiation but have limited barrier effect against UVA radiation.     

EGF-Receptor phosphorylation and signaling are targeted by H2O2 redox stress

Inflammation of the respiratory tract is associated with the production of reactive oxygen species, such as hydrogen peroxide (H2O2) and superoxide (O2-), which contribute extensively to lung injury in diseases of the respiratory tract. The mechanisms and target molecules of these oxidants are mainly unknown but may involve modifications of growth-factor receptors. We have shown that H2O2 induces epidermal growth factor (EGF)-receptor tyrosine phosphorylation in intact cells as well as in membranes of A549 lung epithelial cells. On the whole, total phosphorylation of the EGF receptor induced by H2O2 was lower than that induced by the ligand EGF. Phosphorylation was confined to tyrosine residues and was inhibited by addition of genistein, indicating that it was due to the activation of protein tyrosine kinase (PTK). Phosphoamino acid analysis revealed that although the ligand, EGF, enhanced the phosphorylation of serine, threonine, and tyrosine residues, H2O2 preferentially enhanced tyrosine phosphorylation of the EGF receptor. Serine and threonine phosphorylation did not occur, and the turnover rate of the EGF receptor was slower after H2O2 exposure. Selective H2O2-mediated phosphorylation of tyrosine residues on the EGF receptor was sufficient to activate phosphorylation of an SH2-group-bearing substrate, phospholipase C-gamma (PLC-gamma), but did not increase mitogen-activated protein (MAP) kinase activity. Moreover, H2O2 exposure decreased protein kinase C (PKC)-alpha activity by causing translocation of PKC-alpha from the membrane to the cytoplasm. These studies provide novel insights into the capacity of a reactive oxidant, such as H2O2, to modulate EGF-receptor function and its downstream signaling. The H2O2-induced increase in tyrosine phosphorylation of the EGF receptor, and the receptor's slower rate of turnover and altered downstream phosphorylation signals may represent a mechanism by which EGF-receptor signaling can be modulated during inflammatory processes, thereby affecting cell proliferation and thus having implications in wound repair or tumor formation.     

Impact of ultraviolet irradiation on expression of SSA/Ro autoantigenic polypeptides in transformed human epidermal keratinocytes

SSA/Ro autoantibodies are frequently found in various autoimmune disorders including subacute cutaneous and neonatal lupus erythematosus. SSA/Ro patient sera precipitate a ribonucleoprotein complex consisting of multiple polypeptides and small RNA molecules (hY RNA). Such sera react in Western blot with at least four antigenically distinct proteins having molecular weights of 52-60 kD. Several laboratories have reported increased binding of anti-SSA/Ro patient serum to viable cultured human epidermal keratinocytes following UVB irradiation. However, it is currently unknown which SSA/Ro molecule(s) might be responsible for this increased antibody binding to UVB irradiated keratinocytes. To address this question, we studied the effect of UVB irradiation on the expression of three different polypeptide components of the SSA/Roautoantigen complex (60 kD SSA/Ro, 52 kD SSA/Ro, and 46 kD SSA/Ro (calreticulin) in A431 cells, a transformed human epidermal keratinocytes cell line. Total cellular and cell surface expression of each SSA/Ro antigenic polypeptide was examined by a whole cell ELISA and FACS using rabbit anti-synthetic peptide antisera as probes. Our results suggest that both total cellular and cell surface calreticulin, but not the 60 and 52 kD SSA/Ro polypeptides, is increased after 100 J/M2 of UVB irradiation, indicating that perturbed calreticulin expression may be primarily responsible for the UVB-induced increased binding of anti-SSA/Ro to keratinocytes. These results suggest that calreticulin could be a critical component of the SSA/Ro ribonucleoprotein complex that is involved in the pathogenesis of anti-SSA/Ro-associated photosensitive LE skin lesions.     

(-)-Epigallocatechin-3-gallate inhibition of ultraviolet B-induced AP-1 activity

Green tea polyphenols have been shown to inhibit cancer in a variety of tumor models, including ultraviolet B (UVB)-induced non-melanoma skin cancer. In green tea extracts, the major dry mass constituent is the family of catechins, of which (-)-epigallocatechin-(3)-gallate (EGCG) is considered to be important for the chemopreventive activity. EGCG has been shown to have antioxidant properties, but there has been little progress toward identifying the specific targets and mechanisms of its action. Using cultured human keratinocytes, we show that UVB-induced AP-1 activity is inhibited by EGCG in a dose range of 5.45 nM to 54.5 microM. EGCG is effective at inhibiting AP-1 activity when applied before, after or both before and after UVB irradiation. EGCG also inhibits AP-1 activity in the epidermis of a transgenic mouse model. This work begins to define a mechanism by which EGCG could be acting to inhibit UVB-induced tumor formation.     

Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs

Extensive documentation has validated the role of UV irradiation as a tumor initiator and promoter, inducing both squamous and basal cell carcinomas. Human epidermis is a tissue which undergoes active metabolism of arachidonic acid to prostaglandins which is regulated by the action of prostaglandin H synthase (also known as cyclooxygenase). One mechanism for the promotional activity of UV light may involve its ability to induce prostaglandin formation. Work in our laboratory has demonstrated that acute exposure of human keratinocytes to UVB irradiation results in increased production of prostaglandin E2 (PGE2). When cultured human keratinocytes were examined after irradiation with 30 mJ/cm2 UVB in vitro, Western blot analysis showed a 6-fold increase in COX-2 protein which was evident at 6 h and peaked 24 h after irradiation. Furthermore, when human subjects were irradiated on sun-protected skin with up to four times their minimal erythema dosage (MED) and biopsied 24 h later, upregulation of COX-2 protein expression was observed via immunofluorescence microscopy. RNAase protection assays supported this observation, showing induction of COX-2 message which peaked at approximately 12 h following irradiation in vitro. Furthermore, human squamous cell carcinoma biopsies exhibited strongly enhanced staining for COX-2 protein via immunohistochemistry and Western analysis when compared to normal non-sun-exposed control skin. Together, these data demonstrate acute upregulation of COX-2 via UVB irradiation and suggest the need for further studies of COX-2 expression as a potential pharmacological target mediating human skin tumor development.     

The detailed anatomy of the palmar cutaneous nerves and its clinical implications

We have recently reported that in vitro low dose of ultraviolet B radiation (UVB, 100-200 J per m2) directly impaired the antigen-presenting function of human Langerhans cells. In this study, we analyzed the effect of UVB irradiation on the Langerhans cells expression of several accessory molecules, namely CD54, CD80, and CD86. Langerhans cells phenotype was determined either immediately after UVB exposure (100 J per m2) or after a 2 d culture. No modification in cell surface antigen levels was observed immediately after irradiation. Prior UVB exposure did not modify the levels of CD80 at the Langerhans cells surface after a 2 d culture. In contrast, CD54 and, above all, CD86 expression were significantly decreased. Addition of exogenous anti-CD28 monoclonal antibodies partly restored the allostimulatory property of irradiated Langerhans cells in mixed epidermal cell-lymphocyte reaction, demonstrating that impairment of CD86 upregulation contributes to the UVB-induced immunosuppressive effect. Furthermore, we found that UVB irradiation at 200 J per m2 significantly reduced the number of viable Langerhans cells after 2 d of culture. UVB-induced cytotoxicity was due to apoptotic cell death, as demonstrated by typical morphologic alterations and by DNA fragmentation yielding a classical ladder pattern on gel electrophoresis. Interestingly, interaction of Langerhans cells with CD40-ligand transfected L cells improved the viability of irradiated Langerhans cells, counteracted the inhibition of CD86 expression, and efficiently reduced the number of apoptotic cells after a 2 d culture. Collectively, these results demonstrate that in vitro UVB exposure affects Langerhans cells via at least two distinct pathways: (i) decreased CD86 costimulatory molecule upregulation; and (ii) induction of Langerhans cells apoptosis, a phenomenon partly prevented by CD40 triggering.     

EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL

Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-2 family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), increased the expression of Bcl-XL in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.     

UVB radiation interrupts cytokine-mediated support of an epidermal-derived dendritic cell line (XS52) by a dual mechanism

We have established long-term dendritic cell lines from the epidermis of newborn mice. These cell lines (XS series) proliferate maximally in response to granulocyte/macrophage-colony stimulating factor, as well as to CSF-1, which is produced by skin-derived NS fibroblast lines and by keratinocytes (albeit in smaller amounts). The purpose of this study was to examine the impact of UVB radiation on CSF-1-mediated interaction of dendritic cells with fibroblasts and keratinocytes. Exposure of NS cells to UVB radiation (unfiltered FS20 sunlamp) decreased CSF-1 production at mRNA and protein levels. Both changes occurred in a dose-dependent fashion, with 50 J/m2 causing a significant reduction. UVB radiation also downregulated CSF-1 mRNA expression by Pam 212 keratinocytes. UVB exposure of XS cells diminished the surface expression of CSF-1 receptors, with 50 J/m2 causing a significant reduction. Thus, UVB radiation interrupts CSF-1-mediated cell-cell interaction by a dual mechanism: downregulating CSF-1 production and abrogating CSF-1 receptor expression. Importantly, granulocyte/macrophage-colony stimulating factor receptor expression by XS cells was also inhibited by UVB radiation, once again, with 50 J/m2 producing significant inhibition. We propose that the resulting CSF-1 deficiency in epidermal microenvironment and unresponsiveness by dendritic cells to relevant growth factors may contribute to UVB-mediated loss of resident epidermal dendritic cells (i.e., Langerhans cells) in skin.