TRPV4 Channel-Regulated ATP Release Contributes to γ-Irradiation-Induced Production of IL-6 and IL-8 in Epidermal Keratinocytes.

External stimuli, such as radiation, induce inflammatory cytokine and chemokine production in skin, but the mechanisms involved are not completely understood. We previously showed that the P2Y11 nucleotide receptor, p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) all participate in interleukin (IL)-6 production induced by γ-irradiation. Here, we focused on the transient receptor potential vanilloid 4 (TRPV4) channel, which is expressed in skin keratinocytes and has been reported to play a role in inflammation. We found that irradiation of human epidermal keratinocytes HaCaT cells with 5 Gy of γ-rays (137Cs: 0.75 Gy/min) induced IL-6 and IL-8 production. HaCaT cells treated with TRPV4 channel agonist GSK1016790A also showed increased IL-6 and IL-8 production. In both cases, IL-6/IL-8 production was not increased at 24 h after stimulation, but was increased at 48 h. ATP was released from cells exposed to γ-irradiation or TRPV4 channel agonist, and the release was suppressed by TRPV4 channel inhibitors. The γ-irradiation-induced increase in IL-6 and IL-8 production was suppressed by apyrase (ecto-nucleotidase), NF157 (selective P2Y11 receptor antagonist) and SB203580 (p38 MAPK inhibitor). GSK1016790A-induced inhibitor of kappa B-alpha (IκBα) decomposition, which causes NF-κB activation was suppressed by NF157 and SB203580, and γ-irradiation-induced IκBα decomposition was suppressed by TRPV4 channel inhibitors. Our results suggest that γ-irradiation of keratinocytes induces ATP release via activation of the TRPV4 channel, and then ATP activates P2Y11 receptor and p38 MAPK-NF-κB signaling, resulting in IL-6/IL-8 production.

Effects of Narrow Band Ultraviolet B on Serum Levels of Vascular Endothelial Growth Factor and Interleukin-8 in Patients with Psoriasis.

The aim of this study was to investigate the effects of narrow band ultraviolet B (NB-UVB) therapy on serum levels of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) in patients with psoriasis. Relevant Chinese and English scientific literature databases were searched to identify studies published before November, 2013 that included serum VEGF and IL-8 levels in patients with psoriasis. The studies retrieved from database searches were screened on the basis of predefined selection criteria, and data from finally selected studies were extracted for meta-analysis. Analyses were conducted using STATA 12.0 software. Our systematic search resulted in retrieval of 700 studies (500 studies in Chinese, 200 studies in English), and after a multistep screening process, 13 studies met the inclusion criteria and were enrolled for meta-analysis. The 13 studies contained a combined total of 400 patients with psoriasis and 221 healthy controls. The results of meta-analysis revealed that healthy controls exhibited significantly lower serum level of VEGF, compared to patients with psoriasis before therapy. After NB-UVB, VEGF levels were significantly decreased in patients with psoriasis, as compared to their pretherapy VEGF levels. Although no statistically significant differences were detected in IL-8 serum levels between patients with psoriasis and healthy controls before therapy, after NB-UVB therapy, the serum IL-8 levels in patients with psoriasis were markedly decreased. Corresponding reductions in the psoriasis area and severity index scores of patients with psoriasis were observed after NB-UVB treatment. Our results revealed that NB-UVB therapy significantly decreased the serum levels of VEGF and IL-8 in patients with psoriasis. Furthermore, VEGF and IL-8 levels correlated with disease status, indicating that they are sensitive biomarkers for evaluating the effectiveness of psoriasis therapy.

Cytokine changes in response to radio-/chemotherapeutic treatment in head and neck cancer.

BACKGROUND: Radiation and systemic chemotherapy are standard treatment strategies for advanced or metastatic head and neck cancer. However, little is known about the implications and changes in the tumor microenvironment, including the T-helper (TH)1/TH2 balance in response to these treatment regimens. The aim of the current study was to unravel the effects of chemotherapeutic drugs and radiation on cytokine changes. MATERIALS AND METHODS: In this study, the effect of radiation and chemotherapeutic treatment (5-fluorouracil and cisplatin) on eight cell lines was determined. Before and after exposure, cytokine levels in culture supernatants of cell lines were evaluated using the Bio-Plex Assay (Bio-Rad) and the Human TH1/TH2 Cytometric Bead Array (Becton Dickinson). Results were correlated with parallel measurements for cellular proliferation assessed by cytotoxicity assay. RESULTS: Seven out of eight cell lines of primary tumors or metastases demonstrated an enhanced level of the cytokines interleukin (IL)-1ß, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF) and tumor necrosis factor-α (TNF-α), after sub-lethal radiation doses. Under treatment with low concentrations of 5-fluorouracil and cisplatin, all examined cell lines showed an increasing secretion of the cytokines IL-6 and G-CSF. In contrast, sub-lethal doses of both cytostatic drugs revealed a dose-dependent decrease in secretion IL-1ß. Regarding GM-CSF and TNF-α, we demonstrated an increase in secretion by the primary tumors under low doses of 5-fluorouracil and cisplatin, whereas the metastases showed a sharp drop of GM-CSF and TNF-α secretion. Chemotherapeutic treatment led to no changes of the IL-8 cytokine profile. CONCLUSION: The results suggest complex cytokine changes of the tumor microenvironment and more aberrant expression profiles under treatment with radiation and the chemotherapeutic drugs 5-fluorouracil and cisplatin.

Green light emitting diodes accelerate wound healing: characterization of the effect and its molecular basis in vitro and in vivo.

Because light-emitting diodes (LEDs) are low-coherent, quasimonochromatic, and nonthermal, they are an alternative for low level laser therapy, and have photobiostimulative effects on tissue repair. However, the molecular mechanism(s) are unclear, and potential effects of blue and/or green LEDs on wound healing are still unknown. Here, we investigated the effects of red (638 nm), blue (456 nm), and green (518 nm) LEDs on wound healing. In an in vivo study, wound sizes in the skin of ob/ob mice were significantly decreased on day 7 following exposure to green LEDs, and complete reepithelialization was accelerated by red and green LEDs compared with the control mice. To better understand the molecular mechanism(s) involved, we investigated the effects of LEDs on human fibroblasts in vitro by measuring mRNA and protein levels of cytokines secreted by fibroblasts during the process of wound healing and on the migration of HaCat keratinocytes. The results suggest that some cytokines are significantly increased by exposure to LEDs, especially leptin, IL-8, and VEGF, but only by green LEDs. The migration of HaCat keratinocytes was significantly promoted by red or green LEDs. In conclusion, we demonstrate that green LEDs promote wound healing by inducing migratory and proliferative mediators, which suggests that not only red LEDs but also green LEDs can be a new powerful therapeutic strategy for wound healing.

Development of an in vitro model for radiation-induced effects on oral keratinocytes.

Changes in epithelial cell activity and the production of pro-inflammatory cytokines were examined utilizing an organotypic culture system as an in vitro model to study the effects of radiation on oral keratinocytes to simulate what is thought to occur in radiation-induced oral mucositis. Monolayer cultures of oral keratinocyte were irradiated by varying the dose. Cell injury was assessed using a colony forming efficiency (CFE) assay. Third passage oral keratinocytes were seeded onto AlloDerm to form a 3D construct of an ex vivo produced oral mucosa equivalent (EVPOME) which was irradiated with 0, 1, 3 and 8Gy. Formalin-fixed sections of the EVPOME were used for histology and immunohistochemistry to examine proliferative capacity. Epithelial cell viability of EVPOME was measured by MTT assay. Spent culture medium was used to determine post-radiation pro-inflammatory cytokine production. Basal cells became more swollen and pyknotic as radiation increased, implying loss of cell viability also determined by MTT assay. The number of Ki-67 immunopositive cells and CFE showed negative correlation with radiation, indicating loss of cell proliferative capacity. The production of pro-inflammatory cytokines, IL-1alpha and IL-8, tended to increase in a radiation dose dependent manner. The EVPOME lacking submucosal cellular components was a useful model.

In vitro evaluation of the effects of low-intensity Nd:YAG laser irradiation on the inflammatory reaction elicited by bacterial lipopolysaccharide adherent to titanium dental implants.

BACKGROUND: The bacterial endotoxin lipopolysaccharide (LPS) represents a prime pathogenic factor of peri-implantitis because of its ability to adhere tenaciously to dental titanium implants. Despite this, the current therapeutic approach to this disease remains based mainly on bacterial decontamination, paying little attention to the neutralization of bioactive bacterial products. The purpose of the present study was to evaluate whether irradiation with low-energy neodymium-doped:yttrium, aluminum, and garnet (Nd:YAG) laser, in addition to the effects on bacterial implant decontamination, was capable of attenuating the LPS-induced inflammatory response. METHODS: RAW 264.7 macrophages or human umbilical vein endothelial cells were cultured on titanium disks coated with Porphyromonas gingivalis LPS, subjected or not to irradiation with the Nd:YAG laser, and examined for the production of inflammatory cytokines and the expression of morphologic and molecular markers of cell activation. RESULTS: Laser irradiation of LPS-coated titanium disks significantly reduced LPS-induced nitric oxide production and cell activation by the macrophages and strongly attenuated intercellular adhesion molecule-1 and vascular cell adhesion molecule expression, as well as interleukin-8 production by the endothelial cells. CONCLUSION: By blunting the LPS-induced inflammatory response, Nd:YAG laser irradiation may be viewed as a promising tool for the therapeutic management of peri-implantitis.

Extremely low frequency electromagnetic field enhances human keratinocyte cell growth and decreases proinflammatory chemokine production.

BACKGROUND: Proliferation and differentiation of keratinocytes are central processes in tissue regeneration after injury. Chemokines, produced by a wide range of cell types including keratinocytes, play a regulatory role in inflammatory skin diseases. Several studies have shown that an electromagnetic field (EMF) can influence both inflammatory processes and repair mechanisms including wound healing on different tissue models. OBJECTIVES: To elucidate the effect of extremely low frequency EMF (ELF-EMF) on keratinocyte proliferation and production of chemokines [RANTES, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha and interleukin (IL)-8] in order to evaluate a potential therapeutic use of magnetic fields. METHODS: The human keratinocyte cell line HaCaT was exposed at 1 mT, 50 Hz for different lengths of time and compared with unexposed control cells. Cell growth and viability were evaluated at different exposure times by cell count and trypan blue exclusion. Chemokine production and expression were analysed by enzyme-linked immunosorbent assay (ELISA) and by real-time polymerase chain reaction. Total NF-kappaB p65 was quantified by ELISA. RESULTS: Significantly increased growth rates were observed after 48 h of EMF exposure as compared with control cells, while no difference in cell viabilities were detected. Gene expression and release of RANTES, MCP-1, MIP-1 alpha and IL-8 were significantly reduced after 72 h of exposure. NF-kappaB levels became almost undetectable after only 1 h of EMF exposure, and were inversely correlated with cell density. CONCLUSIONS: Our results show that ELF-EMF modulates chemokine production and keratinocyte growth through inhibition of the NF-kappaB signalling pathway and thus may inhibit inflammatory processes. ELF-EMF could represent an additional therapeutic approach in the treatment of skin injury.

Conjugated linoleic acids modulate UVR-induced IL-8 and PGE2 in human skin cells: potential of CLA isomers in nutritional photoprotection.

Conjugated linoleic acids (CLA), derivatives of linoleic acid found in food products, inhibit chemically induced skin cancers in mice. However, their potential photoprotective properties remain unexplored. We examined whether CLA may modulate ultraviolet radiation (UVR)-induced secretion of interleukin (IL)-8 and prostaglandin E2 (PGE(2)), mediators implicated in UVR-induced inflammation and carcinogenesis, in human skin cells. Since tumour necrosis factor (TNF)-alpha is an early mediator of UVR effects, we also examined influence of CLA on TNF-alpha-induced mediator release. HaCaT keratinocytes were supplemented with CLA isomers cis-9-trans-11 (c9,t11-CLA; > or =90%), trans-10-cis-12 (t10,c12-CLA; > or =90%) or all trans-trans isomers (tt-CLA; 23.7%) in tetrahydrofuran/fetal calf serum (THF/FCS) or THF/FCS control. Supplementation of keratinocytes with c9,t11-CLA reduced Ultraviolet B(UVB)-induced IL-8 from 37 113 +/- 2903 pg/ng protein in control cells to 14 167 +/- 2063 pg/ng protein (P < 0.001). Similarly, t10,c12-CLA reduced UVB-induced IL-8 to 9786 +/- 1291.5 pg/ng protein (P < 0.001). Additionally, t10,c12-CLA and tt-CLA inhibited TNF-alpha-induced IL-8 from 11 669 +/- 1692 pg/ng protein in control cells to 5540 +/- 191 (P < 0.001) and 8082 +/- 1298 pg/ng (P < 0.01) protein, respectively. UVB-induced PGE(2) release was reduced by tt-CLA supplementation, from 4.8 +/- 1.2 to 1.6 +/- 0.8 pg/mg protein (P < 0.01), but increased by t10,c12-CLA to 8.8 +/- 1 pg/mg protein (P < 0.001). Influence of CLA on UVB-induced PGE(2) release was further explored in CCD922SK dermal fibroblasts. CLA isomers reduced UVB-induced PGE(2) in fibroblasts, reaching significance with c9,t11-CLA (98 +/- 5 falling to 0 pg/mg protein, P < 0.05). Hence, CLA isomers differentially modulate UVB effects on skin cells in vitro. CLA-containing foods have potential in photoprotection; the cutaneous effects of individual isomers warrant clinical study.

Identification of the dimerisation interface of human interleukin-8 by IL-8-variants containing the photoactivatable amino acid benzoyl-phenylalanine.

The three-dimensional structure of human interleukin-8 (hIL-8) was determined by the use of NMR and X-ray methodology. At high concentrations interleukin-8 and many other chemokines form a non-covalent homodimer. Several studies have been performed to investigate the relevance of the dimer on receptor activation and led to contradictory results. In order to obtain a better understanding of the dimerisation process, covalently linked homo- and heterodimers were produced by photo-induced dimerisation of hIL-8 analogues that contain the photo-activatable amino acid p-benzoyl-phenylalanine (Bpa) at different positions. Whereas the N-terminal fragment (1-54) was expressed as recombinant thioester, the C-terminal fragments (55-77) that contain Bpa either at position 65 or 74 were obtained by solid-phase peptide synthesis. The segments were combined by expressed protein ligation and led to full length IL-8 variants containing the non-proteinogenic amino acid Bpa at single positions. IP(3) activity tests showed high biological activity for the CXCR1-GFP receptor for both variants comparable to that of the native ligand. The refolded and purified ligation-products were used for dimer formation by UV-irradiation. The analysis of the reaction mixture was performed by gel-electrophoresis and mass spectrometry and showed that dimer formation of IL-8 occurred in a position dependent manner. [Bpa(74)]hIL-8 has a high tendency to form covalent dimers whereas no dimer formation was observed for the variant with Bpa at position 65. Accordingly one residue of the dimerisation interface could be identified.

[The effect of ultraviolet B on interleukin-8 secretion in human keratinocyte cell line].

OBJECTIVE: To investigate the effect of ultraviolet B (UVB) on Interleukin-8 secretion in human keratinocyte cell line. METHODS: The concentration of IL-8 was detected by ELISA kit 24 h after human keratinocytes (HKC) were irradiated by different doses of UVB, and the level of IL-8 was also determined at different times after the same dose of UVB irradiation. RESULTS: The secretion of IL-8 was increased after the HKC were irradiated by UVB, the effect was dose-dependent when UVB ranged from 10 to 40 mJ/cm2, and there was statistically significant difference between the IL-8 level of UVB groups (20-70 mJ/cm2) and the control (0 mJ/cm2) (P<0.01). The level of IL-8 was increased 1 h after the irradiation of 30 mJ/cm2 UVB, and it reached the peak at 12 h. There was statistically significant difference between the IL-8 levels detected at different times (3 h, 6 h, 12 h, 24 h after irradiation) and that at 0 h (P<0.01). CONCLUSION: UVB increase the secretion of IL-8, and the effect is dose-dependent to some extent.

Generation of interleukin 8 in stored apheresis platelet concentrates and the preventive effect of prestorage ultraviolet B radiation.

BACKGROUND: Several recent studies have reported both the generation of cytokines, including interleukin (IL)-1 beta, IL-6, tumor necrosis factor alpha (TNF-alpha), and IL-8, in the supernatants of stored platelet concentrates (PCs) and the implications of this generation in febrile nonhemolytic transfusion reactions. Prestorage filtration is regarded as highly effective in the prevention of cytokine generation. STUDY DESIGN AND METHODS: Studies evaluated 1) the levels of these cytokines in apheresis PCs during storage, 2) the effects of white cell inactivation by ultraviolet B or gamma-radiation on the generation of cytokines, and 3) the effects of poststorage filtration on cytokine levels. The apheresis PCs were treated by either ultraviolet B radiation (20,000 J/m2), gamma-radiation (30 Gy), or filtration. Samples were collected sequentially on various days after storage. Cytokines were determined by enzyme-linked immunosorbent assay. RESULTS: The average white cell count in 15 PCs tested was 2.58 +/- 0.7 x 10(6) per mL (range, 0.7-10 x 10(6)/mL). A detectable level of IL-8 was found at 3 days of storage, and the levels of this cytokine increased progressively with increasing storage time, ranging from 1.6 to 35,280 pg per mL on Day 5 and from 2.7 to 83,601 pg per mL on Day 8. Reverse transcriptase-polymerase chain reaction analysis showed that the level of IL-8 paralleled the expression of IL-8 transcripts. The levels of IL-1 beta, IL-6, TNF-alpha, and monocyte chemotactic protein-1 were very low, even on Day 8. Ultraviolet B-radiated PCs failed to generate IL-8, even at 8 days of storage, whereas levels of IL-8 in gamma-radiated PCs were similar to those in nonirradiated PCs. Poststorage filtration of PCs with a negatively charged polyester filter, but not with a positively charged one, markedly reduced the levels of IL-8. CONCLUSION: Of the cytokines tested, IL-8 had the most evident generation in apheresis PCs during storage. Prestorage inactivation of white cells by ultraviolet B radiation, but not by gamma-radiation, was effective in preventing the generation of cytokines during the storage of PCs.