Expression analysis of box C/D snoRNAs with SNPs between C57BL/6 and MSM/Ms strains in male mouse.

MSM/Ms mouse derived from the Japanese wild mouse has unique characteristics compared to the widely used C57BL/6 mouse. To examine the usefulness of the MSM/Ms mouse for the comparative genomic analysis, expression of small RNAs were analyzed by the large-scale sequence analysis for two strains of mouse, C57BL/6 and MSM/Ms. As a trial, expression of box C/D snoRNAs, which are the most abundant small RNAs in the cell, were analyzed. By the comparison of the read number for each fragment, 11 snoRNAs with single nucleotide polymorphisms (SNPs) were detected. One of the snoRNAs, SNORD53, shows the expression only for MSM/Ms and this snoRNA has a mutation in the box sequence in C57BL/6. Thus, it was demonstrated that the proposed experimental system using SNPs can give new insight for the gene expression regulation.

Genetic profile of adult T-cell leukemia/lymphoma in Okinawa: Association with prognosis, ethnicity, and HTLV-1 strains.

Genetic alterations in adult T-cell leukemia/lymphoma (ATLL), a T-cell malignancy associated with HTLV-1, and their clinical impacts, especially from the perspective of viral strains, are not fully elucidated. We employed targeted next-generation sequencing and single nucleotide polymorphism array for 89 patients with ATLL in Okinawa, the southernmost islands in Japan, where the frequency of HTLV-1 tax subgroup-A (HTLV-1-taxA) is notably higher than that in mainland Japan, where most ATLL cases have HTLV-1-taxB, and compared the results with previously reported genomic landscapes of ATLL in mainland Japan and the USA. Okinawan patients exhibited similar mutation profiles to mainland Japanese patients, with frequent alterations in TCR/NF-ĸB (eg, PRKCB, PLCG1, and CARD11) and T-cell trafficking pathways (CCR4 and CCR7), in contrast with North American patients who exhibited a predominance of epigenome-associated gene mutations. Some mutations, especially GATA3 and RHOA, were detected more frequently in Okinawan patients than in mainland Japanese patients. Compared to HTLV-1-taxB, HTLV-1-taxA was significantly dominant in Okinawan patients with these mutations (GATA3, 34.1% vs 14.6%, P = .044; RHOA, 24.4% vs 6.3%, P = .032), suggesting the contribution of viral strains to these mutation frequencies. From a clinical viewpoint, we identified a significant negative impact of biallelic inactivation of PRDM1 (P = .027) in addition to the previously reported PRKCB mutations, indicating the importance of integrated genetic analysis. This study suggests that heterogeneous genetic abnormalities in ATLL depend on the viral strain as well as on the ethnic background. This warrants the need to develop therapeutic interventions considering regional characteristics.

Identification of genetic alterations in extramammary Paget disease using whole exome analysis.

BACKGROUND: Extramammary Paget disease (EMPD) is a rare cutaneous malignant neoplasm, and the genomic alterations underlying its pathogenesis are unknown. OBJECTIVE: To identify tumor-specific genomic alterations in EMPD. METHODS: Exome analysis was performed in specimens from three EMPD patients, and target amplicon sequencing was done for genes frequently mutated in other adenocarcinomas. RESULTS: Exome analysis revealed recurrent somatic mutations in several genes, includingTP53, PIK3CA, and ERBB2. We identified additional candidate exons by searching the COSMIC database for exons that are frequently mutated in other adenocarcinomas. We obtained 19 exons in 12 genes as candidate exons, and performed target amplicon sequencing in samples obtained from EMPD patients. New somatic mutations in the TP53 gene were identified in six EMPD patients. Single nucleotide polymorphism analysis revealed multiple chromosomal alterations in three EMPD specimens, and two specimens exhibited amplification of chromosome 12p13 and losses of 3p21-24, 7q22 and 13q12-21. CONCLUSION: Our comprehensive genetic analysis identified novel genomic alterations, and will inform treatment options for EMPD.

Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement.

BACKGROUND: Three-dimensional (3D) spheroids are frequently used in toxicological study because their morphology and function closely resemble those of tissue. As these properties are maintained over a long term, repeated treatment of the spheroids with a test object is possible. Generally, in the repeated treatment test to assess cytotoxicity in the spheroids, ATP assay, colorimetric measurement using pigments or high-content imaging analysis is performed. However, continuous assessment of cytotoxicity in the same spheroids using the above assays or analysis is impossible because the spheroids must be disrupted or killed. To overcome this technical limitation, we constructed a simple monitoring system in which cytotoxicity in the spheroids can be continuously monitored by nondestructive bioluminescence measurement. RESULTS: Mouse primary hepatocytes were isolated from transchromosomic (Tc) mice harboring a mouse artificial chromosome (MAC) vector expressing beetle luciferase Emerald Luc (ELuc) under the control of cytomegalovirus immediate early enhancer/chicken ß-actin promoter/rabbit ß-globin intron II (CAG) promoter, and used in 3D cultures. We confirmed that both luminescence and albumin secretion from the spheroids seeded in the 96-well format Cell-ableTM were maintained for approximately 1 month. Finally, we repetitively treated the luminescent 3D spheroids with representative hepatotoxicants for approximately 1 month, and continuously and nondestructively measured bioluminescence every day. We successfully obtained daily changes of the dose-response bioluminescence curves for the respective toxicants. CONCLUSIONS: In this study, we constructed a monitoring system in which cytotoxicity in the same 3D spheroids was continuously and sensitively monitored over a long term. Because this system can be easily applied to other cells, such as human primary cells or stem cells, it is expected to serve as the preferred platform for simple and cost-effective long-term monitoring of cellular events, including cytotoxicity.

Variation in the Effect of Particulate Matter on Pulmonary Function in Schoolchildren in Western Japan and Its Relation with Interleukin-8.

This study aimed to investigate the effects of particulate matter (PM) on pulmonary function in schoolchildren, as well as the relationships of these effects with interleukin-8. Morning peak expiratory flow (PEF) was measured daily in 399 children during April-May 2012, and in 384 of these children during March-May 2013. PEF's association with the daily levels of suspended particulate matter (SPM) and PM < 2.5 mm (PM2.5) was estimated using a linear mixed model. Interleukin-8 promoter activity was assessed in THP-G8 cells stimulated by fallen PM collected at Tottori University Hospital during four periods (two in 2012 and two in 2013). An increase of 14.0 mg/m³ in SPM led to PEF changes of -2.16 L/min in 2012 and -0.81 L/min in 2013, respectively. An increment of 10.7 mg/m³ in PM2.5 was associated with PEF changes of -2.58 L/min in 2012 and -0.55 L/min in 2013, respectively. These associations were only significant in 2012. Interleukin-8 promoter activity was significantly higher in both periods of 2012 than in 2013. There was a significant association between pulmonary function in schoolchildren and daily levels of SPM and PM2.5, but this association may differ depending on the PM's ability to elicit interleukin-8 production.

Difference in interleukin-8 transcriptional activity induced in THP-G8 cells by particulate matter collected in winter and summer in western Japan.

BACKGROUND: Airborne particulate matter (PM) may stimulate production of pro-inflammatory cytokines, and thus exposure to PM affects pulmonary diseases. However, the effects of PM on pulmonary diseases have differed among studies. METHODS: PM was collected from February 2 to 28 (winter) and June 1 to 17 (summer) in 2013 in Yurihama, Japan, using filters to separate particles with different aerodynamic diameters (1.1 to <2.2 µm, 2.2 to <3.3 µm, 3.3 to <7.0 µm, and 7.0 to <10 µm). Interleukin (IL)-8 transcriptional activity in THP-G8 cells was examined in response to winter and summer PM with different diameters. RESULTS: IL-8 transcriptional activity induced by winter PM was significantly higher than that with summer PM for each diameter. For winter PM, IL-8 activity increased with an increased diameter, whereas this activity did not differ between a solvent control and each summer PM diameter. This indicates that summer PM does not stimulate production of IL-8 in THP-G8 cells. CONCLUSION: The effects of PM on production of pro-inflammatory cytokines differ with the season and PM diameter. This suggests that the compositions of PM differ on season, and the evaluation of compositions of PM is important in understanding the association of health with short-term exposure to PM.

Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals.

The Tohoku Medical Megabank Organization reports the whole-genome sequences of 1,070 healthy Japanese individuals and construction of a Japanese population reference panel (1KJPN). Here we identify through this high-coverage sequencing (32.4 × on average), 21.2 million, including 12 million novel, single-nucleotide variants (SNVs) at an estimated false discovery rate of <1.0%. This detailed analysis detected signatures for purifying selection on regulatory elements as well as coding regions. We also catalogue structural variants, including 3.4 million insertions and deletions, and 25,923 genic copy-number variants. The 1KJPN was effective for imputing genotypes of the Japanese population genome wide. These data demonstrate the value of high-coverage sequencing for constructing population-specific variant panels, which covers 99.0% SNVs of minor allele frequency ≥0.1%, and its value for identifying causal rare variants of complex human disease phenotypes in genetic association studies.

Japonica array: improved genotype imputation by designing a population-specific SNP array with 1070 Japanese individuals.

The Tohoku Medical Megabank Organization constructed the reference panel (referred to as the 1KJPN panel), which contains >20 million single nucleotide polymorphisms (SNPs), from whole-genome sequence data from 1070 Japanese individuals. The 1KJPN panel contains the largest number of haplotypes of Japanese ancestry to date. Here, from the 1KJPN panel, we designed a novel custom-made SNP array, named the Japonica array, which is suitable for whole-genome imputation of Japanese individuals. The array contains 659,253 SNPs, including tag SNPs for imputation, SNPs of Y chromosome and mitochondria, and SNPs related to previously reported genome-wide association studies and pharmacogenomics. The Japonica array provides better imputation performance for Japanese individuals than the existing commercially available SNP arrays with both the 1KJPN panel and the International 1000 genomes project panel. For common SNPs (minor allele frequency (MAF)>5%), the genomic coverage of the Japonica array (r(2)>0.8) was 96.9%, that is, almost all common SNPs were covered by this array. Nonetheless, the coverage of low-frequency SNPs (0.5%

Decreased pulmonary function in school children in Western Japan after exposures to Asian desert dusts and its association with interleukin-8.

The objective of the study was to investigate the influence of Asian dust storms (ADS) on pulmonary function of school children and the relationship of this effect with interleukin-8. Morning peak expiratory flow (PEF) was measured daily in 399 children from April to May 2012 and in 384 of these children from March to May 2013. The data were analyzed for an association between ADS events and PEF by linear mixed models. Interleukin-8 transcriptional activity was assessed in THP-G8 cells stimulated by airborne particles collected on ADS days. Seven ADS days were identified: April 23 and 24, 2012; March 8 to 10, 2013; and March 19 and 20, 2013. Changes in PEF after ADS exposure were -8.17 L/min (95% confidence interval, -11.40 to -4.93) in 2012 and -1.17 L/min (-4.07 to 1.74) in 2013, and there was a significant difference between 2012 and 2013. Interleukin-8 transcriptional activity was significantly higher in 2012 at 10.6 ± 2.9-fold compared to 3.7 ± 0.4 in March 8 to 10, 2013, and 2.3 ± 0.2 in March 19 and 20, 2013. The influence of ADS events on pulmonary function of children differs with each ADS event and may be related to interleukin-8 production.

Notch down-regulation in regenerated epidermis contributes to enhanced expression of interleukin-36α and suppression of keratinocyte differentiation during wound healing.

BACKGROUND: Notch signaling controls a number of cellular processes, including cell fate decisions, proliferation, differentiation, and survival/apoptosis, in multiple tissues. In the epidermis, Notch1 functions as a molecular switch that controls the transition of cells from an undifferentiated state into a differentiated state. OBJECTIVE: To clarify the functions of Notch in the regenerated epidermis during wound healing. METHODS: Wounds on mouse skin were immunostained. To investigate the functions of Notch, Notch was inhibited in primary keratinocytes by treatment with a γ-secretase inhibitor and by small interfering RNA-mediated knockdown, and was activated by a recombinant adenovirus approach. RESULTS: Notch1 and Notch2 were down-regulated in the regenerated epidermis during wound healing. To clarify the significance of this down-regulation, we examined its effect on expression of the interleukin (IL)-1 family of proinflammatory cytokines because wounds are exposed to pathogens from the outside world. Among the IL-1 family, IL-36α expression was induced by Notch inhibition. This was consistent with the decreased IL-36α expression in Notch-overexpressing keratinocytes. Notch down-regulation in the regenerated epidermis may reinforce defense against stress from the outside world by inducing IL-36α expression. Next, we examined the effects of Notch down-regulation on keratinocyte growth and differentiation. Notch down-regulation did not alter keratinocyte proliferation. On the other hand, Notch1 down-regulation suppressed induction of spinous layer-specific keratins (keratin1 and keratin10) in keratinocytes, which was consistent with the decreased expression of these keratins in the regenerated epidermis. The reduced levels of these keratins would increase cellular flexibility. CONCLUSION: Notch down-regulation in the epidermis appears to contribute to tissue regeneration during wound healing.

Alarmin function of cathelicidin antimicrobial peptide LL37 through IL-36γ induction in human epidermal keratinocytes.

Several dermatoses, including psoriasis, atopic dermatitis, and rosacea, alter the expression of the innate immune effector human cathelicidin antimicrobial peptide (CAMP). To elucidate the roles of aberrant CAMP in dermatoses, we performed cDNA array analysis in CAMP-stimulated human epidermal keratinocytes, the primary cells responding to innate immune stimuli and a major source of CAMP LL37 in skin. Among LL37-inducible genes, IL-1 cluster genes, particularly IL36G, are of interest because we observed coordinate increases in CAMP and IL-36γ in the lesional skin of psoriasis, whereas virtually no CAMP or IL-36γ was observed in nonlesional skin and normal skin. The production and release of IL-36γ were up to 20-30 ng/ml in differentiated keratinocytes cultured in high-calcium media. G-protein inhibitor pertussis toxin and p38 inhibitor suppressed IL-36γ induction by LL37. As an alarmin, LL37 induces chemokines, including CXCL1, CXCL8/IL8, CXCL10/IP-10, and CCL20/MIP3a, and IL-36 (10-100 ng/ml) augments the production of these chemokines by LL37. Pretreatment with small interfering RNA against IL36γ and IL-36R IL36R/IL1RL2 and IL1RAP suppressed LL37-dependent IL8, CXCL1, CXCL10/IP10, and CCL20 production in keratinocytes, suggesting that the alarmin function of LL37 was partially dependent on IL-36γ and its receptors. Counting on CAMP induction in innate stimuli, such as in infection and wounding, IL-36γ induction by cathelicidin would explain the mechanism of initiation of skin inflammation and occasional exacerbations of psoriasis and skin diseases by general infection.

Validation of multiple single nucleotide variation calls by additional exome analysis with a semiconductor sequencer to supplement data of whole-genome sequencing of a human population.

BACKGROUND: Validation of single nucleotide variations in whole-genome sequencing is critical for studying disease-related variations in large populations. A combination of different types of next-generation sequencers for analyzing individual genomes may be an efficient means of validating multiple single nucleotide variations calls simultaneously. RESULTS: Here, we analyzed 12 independent Japanese genomes using two next-generation sequencing platforms: the Illumina HiSeq 2500 platform for whole-genome sequencing (average depth 32.4×), and the Ion Proton semiconductor sequencer for whole exome sequencing (average depth 109×). Single nucleotide polymorphism (SNP) calls based on the Illumina Human Omni 2.5-8 SNP chip data were used as the reference. We compared the variant calls for the 12 samples, and found that the concordance between the two next-generation sequencing platforms varied between 83% and 97%. CONCLUSIONS: Our results show the versatility and usefulness of the combination of exome sequencing with whole-genome sequencing in studies of human population genetics and demonstrate that combining data from multiple sequencing platforms is an efficient approach to validate and supplement SNP calls.

Effects on asthma and induction of interleukin-8 caused by Asian dust particles collected in western Japan.

OBJECTIVE: Asian dust storms (ADS) contain various airborne particles that may augment airway inflammation by increasing the level of interleukin-8. The objective of the study was to investigate the association of exposure to an ADS with worsening of symptoms of adult asthma and the effect of ADS particles on interleukin-8 transcriptional activity. METHODS: The subjects were 112 patients with mild to moderate asthma who recorded scores for their daily upper and lower respiratory tract symptoms and measured morning peak expiratory flow (PEF) from March to May 2011. Interleukin-8 transcriptional activity was assessed in THP-G8 cells that were exposed to airborne particles collected during days of ADS exposure. RESULTS: Of the 112 patients, 31 had comorbid allergic rhinitis (AR) and/or chronic sinusitis (CS), and had worsened scores for upper respiratory tract symptoms on ADS days compared to non-ADS days. Scores for lower respiratory tract symptoms during ADS days were higher than non-ADS days in all patients. Three patients also had unscheduled hospital visits for exacerbation of asthma on ADS days. However, there was no significant difference in daily morning PEF between ADS and non-ADS days. Airborne particles collected on ADS days induced interleukin-8 transcriptional activity in THP-G8 cells compared to the original soil of the ADS. CONCLUSION: Exposure to an ADS aggravates upper and lower tract respiratory symptoms in patients with adult asthma. ADS airborne particles may increase airway inflammation through enhancement of interleukin-8 transcriptional activity.

In vivo determination of vitamin d function using transgenic mice carrying a human osteocalcin luciferase reporter gene.

Vitamin D is an essential factor for ossification, and its deficiency causes rickets. Osteocalcin, which is a noncollagenous protein found in bone matrix and involved in mineralization and calcium ion homeostasis, is one of the major bone morphogenetic markers and is used in the evaluation of osteoblast maturation and osteogenic activation. We established transgenic mouse line expressing luciferase under the control of a 10-kb osteocalcin enhancer/promoter sequence. Using these transgenic mice, we evaluated the active forms of vitamins D2 and D3 for their bone morphogenetic function by in vivo bioluminescence. As the result, strong activity for ossification was observed with 1 α ,25-hydroxyvitamin D3. Our mouse system can offer a feasible detection method for assessment of osteogenic activity in the development of functional foods and medicines by noninvasive screening.

An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8.

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.

Nickel differentially regulates NFAT and NF-κB activation in T cell signaling.

Nickel is a potent hapten that induces contact hypersensitivity in human skin. While nickel induces the maturation of dendritic cells via NF-κB and p38 MAPK activation, it also exerts immunosuppressive effects on T cells through an unknown mechanism. To elucidate the molecular mechanisms of its effects on T cells, we examined the effects of NiCl(2) on mRNA expression in human CD3+ T cells stimulated with CD3 and CD28 antibodies. Using a DNA microarray and Gene Ontology, we identified 70 up-regulated (including IL-1ß, IL-6 and IL-8) and 61 down-regulated (including IL-2, IL-4, IL-10 and IFN-γ) immune responsive genes in NiCl(2)-treated T cells. The DNA microarray results were verified using real-time PCR and a Bio-Plex(TM) suspension protein array. Suppression of IL-2 and IFN-γ gene transcription by NiCl(2) was also confirmed using Jurkat T cells transfected with IL-2 or IFN-γ luciferase reporter genes. To explore the NiCl(2)-regulated signaling pathway, we examined the binding activity of nuclear proteins to NFAT, AP-1, and NF-κB consensus sequences. NiCl(2) significantly and dose-dependently suppressed NFAT- and AP-1-binding activity, but augmented NF-κB-binding activity. Moreover, NiCl(2) decreased nuclear NFAT expression in stimulated T cells. Using Jurkat T cells stimulated with PMA/ionomycin, we demonstrated that NiCl(2) significantly suppressed stimulation-evoked cytosolic Ca(2+) increases, suggesting that NiCl(2) regulates NFAT signals by acting as a blocker of Ca(2+) release-activated Ca(2+) (CRAC) channels. These data showed that NiCl(2) decreases NFAT and increases NF-κB signaling in T cells. These results shed light on the effects of nickel on the molecular regulation of T cell signaling.

Dual oxidase 1 induced by Th2 cytokines promotes STAT6 phosphorylation via oxidative inactivation of protein tyrosine phosphatase 1B in human epidermal keratinocytes.

Although hydrogen peroxide (H(2)O(2)) is better known for its cytotoxic effects, in recent years it has been shown to play a crucial role in eukaryotic signal transduction. In respiratory tract epithelial cells, the dual oxidase (DUOX) proteins 1 and 2 has been identified as the cellular source of H(2)O(2). However, the expression of DUOX1 or DUOX2 has not yet been examined in keratinocytes. In this study, using a DNA microarray, we demonstrated that, of the seven NOX/DUOX family members in normal human epidermal keratinocytes (NHEK), IL-4/IL-13 treatment augments the expression of only DUOX1 mRNA. We next confirmed the IL-4/IL-13 induction of DUOX1 in NHEK at the mRNA and protein level using quantitative real-time PCR and Western blotting, respectively. In addition, we demonstrated that this augmented DUOX1 expression was accompanied by increased H(2)O(2) production, which was significantly suppressed both by diphenyleneiodonium, an inhibitor of NADPH oxidase, and by small interfering RNA against DUOX1. Finally, we demonstrated that the increased expression of DUOX1 in IL-4/IL-13-treated NHEK augments STAT6 phosphorylation via oxidative inactivation of protein tyrosine phosphatase 1B. These results revealed a novel role of IL-4/IL-13-induced DUOX1 expression in making a positive feedback loop for IL-4/IL-13 signaling in keratinocytes.

Gene expression profiling defines the role of ATP-exposed keratinocytes in skin inflammation.

BACKGROUND: Various environmental stimuli, e.g., mechanical stress, osmolarity change, oxidative stress, and microbial products trigger ATP release from cells. It is well known that ATP regulates cell growth, differentiation, terminal differentiation, and cell-to-cell communication in keratinocytes. Moreover, extracellular ATP stimulates the expression and release of IL-6 and modulates the production several chemokines by keratinocytes. OBJECTIVE: To investigate the role of ATP-stimulated keratinocytes in skin inflammation and immune response. METHODS: We identified genes whose expression is augmented in ATP-stimulated human keratinocytes by DNA microarray. These microarray data were validated by quantitative real-time RT-PCR. Furthermore, we confirmed the observed mRNA change at protein level by ELISA and Western blotting. RESULTS: The statistical analysis of the microarray data revealed that, besides IL-6, the expression of several novel genes such as IL-20, CXCL1-3, and ATF3 was significantly augmented in ATP-stimulated keratinocytes. These data was validated by quantitative real-time RT-PCR. We also confirmed the augmented production of IL-6, IL-20, CXCL1 by ELISA and that of ATF3 by Western blotting. Since both IL-6 and IL-20 that can stimulate STAT3 were produced by the ATP-stimulated keratinocytes, we examined their phosphorylation of STAT3. The study demonstrated biphasic activation of STAT3 after ATP stimulation, which was composed of a first peak at 1-2 h and a second peak at 12-24 h. The latter peak was significantly suppressed by anti-IL-6 antibody. CONCLUSION: These studies characterized (1) STAT3 activation, (2) chemotaxis for neutrophils via CXCL1-3, and (3) ATF3 activation as possible roles of ATP-stimulated keratinocytes in skin inflammation and immune response.