In Situ Action Spectra Suggest that DNA Damage is Involved in Ultraviolet Radiation-induced Immunosuppression in Humans

Abstract— The mixed epidermal cell lymphocyte reaction (MECLR) is a commonly used method to study the immunomodulatory effects of UV radiation. The in vitro action spectrum for the MECLR showed that the UV-induced suppression of the MECLR responses is associated with UV-induced DNA damage. To investigate whether in vivo DNA damage also leads to the abrogation of the MECLR, in situ action spectra were made for the MECLR and the induction of thymine dimers (T<>T). Human skin, obtained from plastic surgery, was exposed to monochromatic light of 254, 297, 302 and 312 nm. After irradiation, epidermal cells were isolated and used as stimulator cells in the MECLR or processed for flow cytometric detection of T<>T. On the basis of dose-response curves for each wavelength, the action spectra for suppression of the MECLR and the induction of T<>T were calculated. These spectra showed close similarities, suggesting that, also in situ, UV-induced DNA damage is involved in the UV-induced suppression of the MECLR. Both action spectra showed a small decline from 254 nm to 302 nm, followed by a steep decline to 312 nm. These data show that, in situ, UVC can efficiently induce DNA damage and modulate cutaneous immune responses.     

Ultraviolet radiation attenuates thrombospondin 1 expression via PI3K-Akt activation in human keratinocytes

Thrombospondin 1 (TSP1) is an extracellular glycoprotein and a recognized inhibitor of angiogenesis. Recent studies have demonstrated that UV radiation induces an angiogenic switch, by which it alters the balance between pro- and anti-angiogenic factors in the skin. Here we describe the effects of acute UV exposure on TSP1 expression in human skin epidermis, primary keratinocytes and the epidermal cell line HaCaT. We found that protein and mRNA expressions of TSP1 are significantly reduced in human skin in vivo and in keratinocytes in vitro by a single UV exposure. In human skin and keratinocytes, UV exposure induced the phosphorylation of Akt, a downstream target of the PI3K pathways. Specific inhibitors of PI3K, wortmannin and LY294002, completely blocked Akt activation and UV-induced TSP1 downregulation in keratinocytes. We showed that a specific Akt phosphorylation inhibitor and small interfering RNA-mediated Akt depletion were also blocked by UV-induced TSP1 downregulation in keratinocytes. In conclusion, our findings demonstrate that acute UV exposure downregulates TSP1 expression via PI3K-Akt activation in human keratinocytes. These novel findings may help us understand the regulatory mechanisms of UV-induced skin angiogenesis.     

DNA damage, apoptosis and langerhans cells--Activators of UV-induced immune tolerance

Solar UVR is highly mutagenic but is only partially absorbed by the outer stratum corneum of the epidermis. UVR can penetrate into the deeper layers of the epidermis, depending on melanin content, where it induces DNA damage and apoptosis in epidermal cells, including those in the germinative basal layer. The cellular decision to initiate either cellular repair or undergo apoptosis has evolved to balance the acute need to maintain skin barrier function with the long-term risk of retaining precancerous cells. Langerhans cells (LCs) are positioned suprabasally, where they may sense UV damage directly, or indirectly through recognition of apoptotic vesicles and soluble mediators derived from surrounding keratinocytes. Apoptotic vesicles will contain UV-induced altered proteins that may be presented to the immune system as foreign. The observation that UVR induces immune tolerance to skin-associated antigens suggests that this photodamage response has evolved to preserve the skin barrier by protecting it from autoimmune attack. LC involvement in this process is not clear and controversial. We will highlight some basic concepts of photobiology and review recent advances pertaining to UV-induced DNA damage, apoptosis regulation, novel immunomodulatory mechanisms and the role of LCs in generating antigen-specific regulatory T cells.     

Prevention of UV radiation-induced premature skin aging in hairless mice by the novel compound Melanocin A

Repetitive exposure of the skin to UV radiation induces various harmful changes, such as thickening, wrinkle formation, inflammation and carcinogenesis. A variety of natural compounds and synthetic compounds have been studied to determine whether they can prevent UV-induced harmful effects. In this study, we investigated the effect of a novel compound, Melanocin A, which was isolated from Eupenicillium shearii F80695, on UV-induced premature skin aging. First, we studied the effect of Melanocin A on UV-induced matrix metalloproteinase (MMP)-9 expression in an immortalized human keratinocyte cell line, HaCaT, in vitro. Acute UV irradiation induced MMP-9 expression at both the mRNA and protein levels and Melanocin A suppressed this expression in a dose-dependent manner. We then investigated the effect of Melanocin A on UV-induced skin changes in hairless mice in vivo. Chronic exposure of hairless mouse dorsal skin to UV increased skin thickness and induced wrinkle formation and the gelatinase activities of MMP-2 and MMP-9. Moreover, Melanocin A significantly suppressed UV-induced morphologic skin changes and MMP-2 and MMP-9 expression. Taken together, these results show that Melanocin A can prevent the harmful effects of UV that lead to skin aging. Therefore, we suggest that Melanocin A should be viewed as a potential therapeutic agent for preventing and/or treating premature skin aging.     

A re-evaluation of the role of host defence peptides in mammalian immunity

Host defence peptides are found in all classes of life and are a fundamental component of the innate immune response. Initially it was believed that their sole role in innate immunity was to kill invading microorganisms, thus providing direct defence against infection. Evidence now suggests that these peptides play diverse and complex roles in the immune response and that, in higher animals, their functions are not restricted to the innate immune response. In in vitro experiments certain host defence peptides have been demonstrated to be potent antimicrobial agents at modest concentrations, although their antimicrobial activity is often strongly reduced or ablated in the presence of physiological concentrations of ions such as Na(+) and Mg(2+). In contrast, in experiments done in standard tissue culture media, the composition of which more accurately represents physiological levels of ions, mammalian host defence peptides have been demonstrated to have a number of immunomodulatory functions including altering host gene expression, acting as chemokines and/or inducing chemokine production, inhibiting lipopolysaccharide induced pro-inflammatory cytokine production, promoting wound healing, and modulating the responses of dendritic cells and cells of the adaptive immune response. Animal models indicate that host defence peptides are crucial for both prevention and clearance of infection. As interest in the in vivo functions of host defence peptides is increasing, it is important to consider whether in mammals the direct antimicrobial and immunomodulatory properties observed in vitro are physiologically relevant, especially since many of these activities are concentration dependent. In this review we summarize the concentrations of host defence peptides and ions reported throughout the body and compare that information with the concentrations of peptides that are known have antimicrobial or immunomodulatory functions in vitro.     

Purification and Identification of Novel Myeloperoxidase Inhibitory Antioxidant Peptides from Tuna (Thunnas albacares) Protein Hydrolysates

Antioxidative peptides that inhibit myeloperoxidase (MPO) enzyme activity can effectively defend against oxidative stress damage. The antioxidant peptides from tuna protein were produced using alcalase hydrolysis and purified by ultrafiltration and Sephadex G-15, and the fractions with the highest free radicals scavenging ability and oxygen radical absorbance capacity (ORAC) values were sequenced using HPLC–MS/MS. Fifty-five peptide sequences were identified, 53 of which were successfully docked into MPO. The representative peptide ACGSDGK had better antioxidant activity and inhibition of MPO chlorination and peroxidation than the reference peptide hLF1-11. The docking model further showed intense molecular interactions between ACGSDGK and MPO, including hydrogen bonds, charge, and salt bridge interactions, which occluded the active site and blocked the catalytic activity of MPO. These results suggested that the antioxidant peptide ACGSDGK has the potential to inhibit oxidative stress and alleviate inflammation in vivo because of its inhibitory effect on the MPO enzyme.     

Bradykinin-related peptides from Phyllomedusa hypochondrialis azurea: Mass spectrometric structural characterisation and cloning of precursor cDNAs

Amphibian skin secretions contain a plethora of bioactive compounds, many of which are understood to act to deter ingestion by predators. Bradykinins in particular are constitutively expressed in many amphibian skin secretions, mediating a variety of effects including hyperalgesia and contraction of gastric smooth muscle. Using a variety of proteomic techniques (high-performance liquid chromatography (HPLC) separation, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), and quadrupole time-of-flight tandem mass spectrometry (Q-TOF-MS/MS)) the current study identified 13 bradykinin-like peptides in the skin secretions of Phyllomedusa hypochondrialis azurea, including several new C-terminally extended isoforms (VPPGFTPFRLT, VHypPGFTPFRQT) and a novel phyllokinin-like peptide (RPPGFTPFRVY). Identification of the cDNA sequences encoding these peptides led to the deduction that the peptides were derived from differential post-translational processing and modification of five different precursors. Such an event emphasises the metabolic efficiency of peptide production in amphibian venom, with multiple products perhaps selective to different receptors in a variety of predators generated from a single precursor. An unusual modification was also recognised in the present study, with several bradykinin-like peptides featuring hydroxyprolination of the first proline residue rather than the commonly targeted second. This alteration may be mediated by the structural organisation of N-terminal amino acids prior to precursor processing.     

In vivo targeting of organic calcium sensors via genetically selected peptides

A library of constrained peptides that form stable folded structures was screened for aptamers that bind with high affinity to the fluorescent dye Texas red. Two selected clones had binding constants to Texas red of 25 and 80 pM as phage and binding had minimal effects on the fluorescence of Texas red. The peptides interact with distinct but overlapping regions of Texas red. One peptide bound to X-rhod calcium sensors, which share the same core fluorophore as Texas red. These dyes retained calcium sensitivity when bound to the peptide. This peptide was used to label a fusion protein with X-rhod-5F in vivo, and X-rhod sensed changes in calcium locally. Thus, minimal, constrained peptides can functionally bind to environmentally sensitive dyes or other organic agents in biological contexts, suggesting tools for in vivo imaging and analysis.     

UV-induced immunosuppression in the balance

Around 1980, experiments with hairless mice showed us that UV-induced actinic keratoses (AK) and ensuing skin carcinomas did not arise independently: the rate of occurrence in one skin area was increased considerably if AKs had already been induced separately in another distant skin area, i.e. a systemic effect. The ground laying work of Margaret Kripke in the 1970s provided a fitting explanation: UV-induced immunosuppression and tolerance toward the UV-induced tumors. From Kripke's work a new discipline arose: "Photoimmunology." Enormous strides were made in exploring and expanding the effects from UV carcinogenesis to infectious diseases, and in elucidating the mechanisms involved. Stemming from concerns about a depletion of the ozone layer and the general impact of ambient UV radiation, the groups I worked in and closely collaborated with explored the anticipated adverse effects of UV-induced immunosuppression on healthy individuals. An important turning point was brought about in 1992 when the group of Kevin Cooper reported that immunosuppression could be induced by UV exposure in virtually all human subjects tested, suggesting that this is a normal and sound physiological reaction to UV exposure. This reaction could actually protect us from illicit immune responses against our UV-exposed skin, such as observed in idiopathic polymorphic light eruption. This premise has fruitfully rekindled the research on this common "sun allergy," affecting to widely varying degrees about one in five Europeans with indoor professions.     

The impact of impurities in synthetic peptides on the outcome of T-cell stimulation assays

Protein-spanning peptide pools have proven valuable as a screening tool for detecting T-lymphocyte responses against a wide range of proteins. We have used this approach in our search for T cells reactive to the onconeural protein HuD. We found positive responses in only 3 of 127 individuals; however, these were highly unusual in that the same class I HLA alleles and peptides were involved. These T-cell responses were not confirmed when peptides re-synthesized by the same manufacturer with similar and with higher purity levels were used. Our observations indicated that these T-cell responses were not directed against the designed HuD peptides. Here, we report on (i) comparisons of the peptide batches analyzed by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) that did--and did not--elicit T-cell responses and (ii) a detailed analysis of the various by-products of peptides, irrespective of T-cell assay outcome. We found numerous differences between the peptide batches, such as omissions of amino acids in the primary structure of the peptides. Furthermore, some batches revealed strong interactions with calcium ions or contained sulfated peptides. Our data reveal that different batches from the same peptide may contain artefacts that influence the outcome of HLA-restricted T-cell response assays.     

A critical role for dermal mast cells in cis-urocanic acid-induced systemic suppression of contact hypersensitivity responses in mice

Many studies have implicated cis-urocanic acid (cis-UCA) in UVB-induced immunomodulation. The strongest evidence came from studies in mice whereby a cis-UCA antibody blocked UVB-induced suppression of delayed-type hypersensitivity responses. Furthermore, in several studies, the cis-UCA antibody at least partially reversed UVB suppression of contact hypersensitivity responses. Previous reports suggested that cis-UCA was immunomodulatory through its effects on keratinocytes, Langerhans cells, fibroblasts, T lymphocytes, natural killer cells and monocytes/macrophages. As dermal mast cells were recently demonstrated to be critical to UVB-induced systemic suppression of certain delayed-type and contact hypersensitivity responses, we investigated whether they were involved in the processes by which cis-UCA was immunomodulatory. Not only was there a correlation between dermal mast cell prevalence and the degree of susceptibility of different strains of mice to the immunomodulatory effects of cis-UCA, there was also a functional link. Mast cell-depleted Wf/Wf mice were rendered susceptible to immunomodulation by cis-UCA injected subcutaneously only after their dorsal skin had been reconstituted with bone marrow-derived mast cell precursors. These studies suggest that mast cells are critical to the processes by which cis-UCA suppresses systemic contact hypersensitivity responses to the hapten, trinitrochlorobenzene, in mice.     

Rational design and molecular diversity for the construction of anti-alpha-bungarotoxin antidotes with high affinity and in vivo efficiency

The structure of peptide p6.7, a mimotope of the nicotinic receptor ligand site that binds alpha-bungarotoxin and neutralizes its toxicity, was compared to that of the acetylcholine binding protein. The central loop of p6.7, when complexed with alpha-bungarotoxin, fits the structure of the acetylcholine binding protein (AChBP) ligand site, whereas peptide terminal residues seem to be less involved in toxin binding. The minimal binding sequence of p6.7 was confirmed experimentally by synthesis of progressively deleted peptides. Affinity maturation was then achieved by random addition of residues flanking the minimal binding sequence and by selection of new alpha-bungarotoxin binding peptides on the basis of their dissociation kinetic rate. The tetra-branched forms of the resulting high-affinity peptides were effective as antidotes in vivo at a significantly lower dose than the tetra-branched lead peptide.     

Peptide phage display as a tool for drug discovery: targeting membrane receptors

Ligands selected from phage-displayed random peptide libraries tend to be directed to biologically relevant sites on the surface of the target protein. Consequently, peptides derived from library screenings often modulate the target protein's activity in vitro and in vivo and can be used as lead compounds in drug design and as alternatives to antibodies for target validation in both genomics and drug discovery. This review discusses the use of phage display to identify membrane receptor modulators with agonistic or antagonistic activities. Because isolating or producing recombinant membrane proteins for use as target molecules in library screening is often impossible, innovative selection strategies such as panning against whole cells or tissues, recombinant receptor ectodomains, or neutralizing antibodies to endogenous binding partners were devised. Prominent examples from a two-decade history of peptide phage display will be presented, focusing on the design of affinity selection experiments, methods for improving the initial hits, and applications of the identified peptides.     

Nicotinamide Prevents Ultraviolet Radiation-induced Cellular Energy Loss

UV radiation is carcinogenic by causing mutations in the skin and also by suppressing cutaneous antitumor immunity. We previously found nicotinamide (vitamin B3) to be highly effective at reducing UV-induced immunosuppression in human volunteers, with microarray studies on in vivo irradiated human skin suggesting that nicotinamide normalizes subsets of apoptosis, immune function and energy metabolism-related genes that are downregulated by UV exposure. Using human adult low calcium temperature keratinocytes, we further investigated nicotinamide’s effects on cellular energy metabolism. We found that nicotinamide prevented UV-induced cellular ATP loss and protected against UV-induced glycolytic blockade. To determine whether nicotinamide alters the effects of UV-induced oxidative stress posttranslationally, we also measured UV-induced reactive oxygen species (ROS). Nicotinamide had no effect on ROS formation, and at the low UV doses used in these studies, equivalent to ambient daily sun exposure, there was no evidence of apoptosis. Hence, nicotinamide appears to exert its UV protective effects on the skin via its role in cellular energy pathways.     

Role of UV in cutaneous melanoma

Malignant melanoma arises from epidermal melanocytes, the cells responsible for the production of the skin pigment melanin. The photoprotective role of melanin, which is transferred to neighboring keratinocytes, in UV-induced skin carcinogenesis, specifically in nonmelanoma skin cancers, has been well documented. Although melanocyte-resident melanin is expected to offer similar protection to melanocytes from UV-induced damage, UV radiation has long been suspected to have an etiologic role in cutaneous melanoma. However, nearly three decades of efforts using a variety of in vitro and in vivo models of human skin and mouse genetic models have produced conflicting data. Epidemiologic studies have also failed to establish a definitive association between UV exposure and risk of melanoma. In this review, we evaluate the dual role of the melanin pigment as a photoprotector as well as a photosensitizer and examine the evidence for association between melanin levels (constitutive and induced) and melanoma risk. We also discuss possible reasons for the lack of signature UV mutations in melanoma oncogenes known to date and potential alternative mechanisms to explain the role of UV in melanomagenesis.     

螺旋型抗菌肽的疏水性对抗细菌与抗真菌活性的影响比较

α-螺旋型多肽HPRP-A1由15个氨基酸残基组成,来源于幽门螺杆菌核糖体蛋白L1的N端.本研究以HPRP-A1为模板,在其非极性面中心通过单个氨基酸定点取代的方法,形成一系列疏水性不同的多肽类似物,系统地研究疏水性对α-螺旋型多肽生物活性的影响.结果显示,多肽疏水性及所带净电荷对多肽生物活性起着重要的作用;HPRP-A1及疏水性相对较高的多肽类似物具有较好的广谱抗菌活性（包括革兰氏阳性菌、革兰氏阴性菌及真菌）,但也有相对较高的溶血活性;多肽的疏水性与所带净电荷的变化对多肽抗细菌活性与抗真菌活性所产生的影响有着相似的变化趋势和程度.这意味着多肽与细菌的作用机制和多肽与真菌的作用机制存在一定的相关性.多肽对细菌和真菌的抗菌活性存在特异性,为设计出具有临床应用前景的抗菌肽药物奠定了基础.     

Comparison on effect of hydrophobicity on the antibacterial and antifungal activities of α-helical antimicrobial peptides

HPRP-A1, a 15-mer α-helical cationic peptide, was derived from N-terminus of ribosomal protein L1 (RpL1) of Helicobacter pylori. In this study, HPRP-A1 was used as a framework to obtain a series of peptide analogs with different hydrophobicity by single amino acid substitutions in the center of nonpolar face of the amphipathic helix in order to systematically study the effect of hydrophobicity on biological activities of -helical antimicrobial peptides. Hydrophobicity and net charge of peptides played key roles in the biological activities of these peptide analogs; HPRP-A1 and peptide analogs with relative higher hydrophobicity exerted broad spectrum antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria and pathogenic fungi, but also showed stronger hemolytic activity; the change of hydrophobicity and net charge of peptides had similar effects with close trend and extent on antibacterial activities and antifungal activities. This indicated that there were certain correlations between the antibacterial mode of action and the antifungal mode of action of these peptides in this study. The peptides exhibited antimicrobial specificity for bacteria and fungi, which provided potentials to develop new antimicrobial drugs for clinical practices.     

Silymarin, a flavonoid from milk thistle (Silybum marianum L.), inhibits UV-induced oxidative stress through targeting infiltrating CD11b+ cells in mouse skin

Phytochemicals have shown promise in inhibiting UV-induced oxidative stress, and therefore are considered as potent inhibitors of UV-induced oxidative stress-mediated skin diseases. We have shown previously that topical treatment of silymarin, a flavonoid from milk thistle (Silybum marianum), inhibits UV-induced oxidative stress in mouse skin. However, the cellular targets responsible for the inhibition of UV-induced oxidative stress by silymarin are not clearly defined. To address this issue, C3H/HeN mice were UV irradiated (90 mJ cm(-2)) with or without topical treatment with silymarin (1 mg cm(-2) skin area). Mice were killed 48 h later and skin samples collected. Flow cytometric analysis of viable dermal cells revealed that the number of infiltrating CD11b+ cells were the major source of oxidative stress (31.8%) in UV-irradiated skin compared with non-UV-exposed skin (0.4%). Treatment of silymarin inhibited UV-induced oxidative stress through inhibition of infiltrating CD11b+ cells. The analysis of myeloperoxidase also indicated that silymarin significantly (P < 0.001) decreased UV-induced infiltration of leukocytes, and this effect of silymarin was similar to that of intraperitoneal treatment of mice with monoclonal antibodies to CD11b. The inhibitory effect of silymarin, regardless of whether it is topically treated before or after UV irradiation, was of similar magnitude. Intraperitoneal administration of monoclonal antibodies to CD11b (rat IgG2b) to C3H/HeN mice inhibited UVB-induced oxidative stress generated by both epidermal and dermal cells as is evident by relative fluorescence intensity of oxidized rhodamine. Similar to the effect of anti-CD11b, silymarin also inhibited UV-induced oxidative stress in both epidermal and dermal cells. Further, CD11b+ and CD11b- cell subsets from UV-treated or silymarin+UV-treated mice were separated by immunomagnetic cell isolation technique from total epidermal and dermal single cell suspensions and analyzed for reactive oxygen species (ROS)/H2O2 production. Analytic data revealed that CD11b+ cell population from UV-irradiated skin resulted in significantly higher production of ROS in both epidermis and dermis than CD11b- cell population, and that silymarin inhibited UV-induced oxidative stress through targeting infiltrating the CD11b+ cell type in the skin.