The leukotriene B4 /BLT1-dependent neutrophil accumulation exacerbates immune complex-mediated glomerulonephritis.

Crescent formation is the most important pathological finding that defines the prognosis of nephritis. Although neutrophils are known to play an important role in the progression of crescentic glomerulonephritis, such as anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis, the key chemoattractant for neutrophils in ANCA-associated glomerulonephritis has not been identified. Here, we demonstrate that a lipid chemoattractant, leukotriene B4 (LTB4 ), and its receptor BLT1 are primarily involved in disease pathogenesis in a mouse model of immune complex-mediated crescentic glomerulonephritis. Circulating neutrophils accumulated into glomeruli within 1 h after disease onset, which was accompanied by LTB4 accumulation in the kidney cortex, leading to kidney injury. LTB4 was produced by cross-linking of Fc gamma receptors on neutrophils. Mice deficient in BLT1 or LTB4 biosynthesis exhibited suppressed initial neutrophil infiltration and subsequent thrombotic glomerulonephritis and renal fibrosis. Depletion of neutrophils before, but not after, disease onset prevented proteinuria and kidney injury, indicating the essential role of neutrophils in the early phase of glomerulonephritis. Administration of a BLT1 antagonist before and after disease onset almost completely suppressed induction of glomerulonephritis. Finally, we found that the glomeruli from patients with ANCA-associated glomerulonephritis contained more BLT1-positive cells than glomeruli from patients with other etiologies. Taken together, the LTB4 -BLT1 axis is the key driver of neutrophilic glomerular inflammation, and will be a novel therapeutic target for the crescentic glomerulonephritis.

Best practice transfer by public health nurses in Japan: actual conditions and related factors.

BACKGROUND: The workload of public health nurses (PHNs) working for local governments has been increasing as health issues become more diverse and complicated. Even amidst the ongoing administrative and fiscal reforms, there is an urgent need to ensure how effectively and efficiently public health nurses can practice in health service development. The objective of this research was to clarify the actual conditions of best practice transfer (BPT) and its related factors. METHODS: An anonymous postal and self-administered questionnaire survey was conducted among PHNs working at 334 sites, including the local government offices and health centers across Japan, and analysed mainly through logistic regression analysis. RESULTS: One hundred eighty-five of the 334 institutions (55.4%) agreed to participate, and of the 966 questionnaire forms distributed, 709 forms (73.4%) were collected, of which 702 responses (72.7%) were valid. Although less than half (43.2%) have experience in BPT in health service development, more than 80% are willing to perform going forward. Significant factors for both the group with experience in BPT and the group with willingness to perform include an organizational culture that promotes BPT, as well as multiple elements of the workplace environment and facilitating factors related to knowledge and learning. The experienced group recognised the needs for criteria to evaluate the adaptability of best practice, while the willing group, to evaluate the quality of practice. CONCLUSIONS: Through a nationwide survey, this research elucidated for the first time the actual conditions of BPT by PHNs in Japan and related factors. The results indicated the importance of developing a system to promote BPT at the workplace level, also highlighted the importance for practitioners and experts, including researchers, to work together to develop practical guidelines to ensure evidence-based practices. Urgent actions are needed for the national and local governments to develop a system to promote BPT from diverse perspectives, building on the findings of this research.

Deficiency of leukotriene B4 receptor type 1 ameliorates ovalbumin-induced allergic enteritis in mice.

Leukotriene B4 receptor type 1 (BLT1), a high-affinity receptor for leukotriene B4 (LTB4), plays an important role in inflammatory responses, including allergic airway inflammation. In this study, we examined the effect of genetic BLT1 deletion (BLT1KO) on ovalbumin (OVA)-induced allergic enteritis in mice to determine the pathogenic role of LTB4/BLT1 in allergic enteritis, a gastrointestinal form of food allergy. Repeated oral OVA challenges after sensitization with OVA and aluminium potassium sulphate induced allergic enteritis, characterized by systemic allergic symptoms (scratching, immobility and swelling), diarrhoea, colonic oedema and colonic goblet cell hyperplasia, accompanied by increased colonic peroxidase activity, colonic inflammatory cytokine expression and increased serum OVA-specific IgE levels. The severity of enteritis was significantly attenuated in BLT1KO mice compared with wild-type (WT) mice, without an increase in serum OVA-specific IgE levels. The accumulation of neutrophils, eosinophils, M2-macrophages, dendritic cells, CD4+ T cells and mast cells was observed in the colonic mucosa of allergic enteritis, and such accumulation was significantly lower in BLT1KO mice than in WT mice. BLT1 expression was upregulated and colocalized mostly in neutrophils and partly in eosinophils and dendritic cells in the colonic mucosa of allergic enteritis. These findings indicate that BLT1 deficiency ameliorates OVA-induced allergic enteritis in mice and that LTB4/BLT1 contributes to neutrophil and eosinophil accumulation in the allergic colonic mucosa. Therefore, BLT1 is a promising drug target for treating food allergies.

[Developing scales to evaluate community practices among municipal public health nurses: content, perceptions of public health nurses, and organizational environment].

Objectives　This study aimed to develop scales for evaluating the level of implementation of community practices and explore the perception of public health nurses in municipalities regarding their community practices.Methods　Draft scales were developed based on a literature review and interviews with municipal public health nurses. Subsequently, a questionnaire survey was conducted with municipal public health nurses across Japan in response to these draft scales. Respondents were recruited based on the municipality population size. Questionnaires were distributed to 2,074 individuals from 52 municipalities ensuring representation from diverse municipalities. Exploratory factor analysis was performed to determine the number of factors, while confirmatory factor analysis was performed to determine the factor structure. The reliability of the scales was assessed by calculating Cronbach's alpha coefficients, and their validity was tested by examining correlations with existing scales, namely the Moral Competence Scale for Public Health Nurses and the Professional Identity Scale for Public Health Nurses, and years of experience as a public health nurse.Results　A total of 721 (34.8%) valid respondents were included in the analysis. Three scales, methods of community practices (three factors, nine items), perceptions of public health nurses toward community residents through community practices (three factors, 10 items), and organizational environment supporting community practices (two factors, 11 items) were developed as indicators of the implementation of public health nurses' community practices. Cronbach's alpha coefficients for the three scales were 0.896, 0.913, and 0.868, respectively. As hypothesized, each subfactor exhibited a positive correlation with the existing scales. However, certain subfactors did not demonstrate any correlation with years of experience.Conclusion　The three scales developed in this study were individually examined for reliability and validity. These scales can be used independently or in combination, allowing public health nurses to select the most suitable scale(s) based on their objective.　A notable contribution of this study is the establishment of concrete indicators for evaluating community practices, addressing the previously vague nature of this evaluation. By incorporating the scale items into daily health practices, we anticipate that these indicators can be employed to evaluate community practices at the organizational and individual levels.

The c-terminal region of BLT2 restricts its localization to the lateral membrane in a LIN7C-dependent manner.

Leukotriene B4 receptor type 2 (BLT2) is a G protein-coupled receptor (GPCR) mainly expressed in epithelial cells, where it enhances barrier function. A unique characteristic of BLT2 is its restricted localization to the lateral membrane. However, the molecular mechanism underlying the localization of BLT2 to the lateral membrane and the physiological roles of laterally localized BLT2 are unknown. BLT1 is the most homologous GPCR to BLT2 and localizes to both the apical and lateral membranes. In this study, we generated chimeric receptors of BLT2 and BLT1 as well as deletion mutants of BLT2 to determine the region(s) of BLT2 responsible for its localization. Chimeric receptors containing the C-terminal domain of BLT2 localized only to the lateral membrane, and the C-terminal deletion mutant of BLT2 accumulated at the Golgi apparatus. Furthermore, the middle and C-terminal regions of BLT2 were important for maintaining epithelial barrier function. Proteomics analysis using the chimeric BLT-ascorbate peroxidase 2 biotinylation method showed that some proteins involved in intracellular protein transport, cell-cell junctions, and actin filament binding were located very close to the C-terminal domain of BLT2. Knockdown of lin-7 homolog C (LIN7C), a membrane trafficking protein, led to accumulation of BLT2 in the Golgi apparatus, resulting in diminished epithelial barrier function. These results suggest that the C-terminal region of BLT2 plays an important role in the transport of BLT2 from the Golgi apparatus to the plasma membrane in a LIN7C-dependent manner.

Novice public health nurses' home-visit skills in Japan: A one-year longitudinal study.

OBJECTIVE: This study aimed to clarify the status of novice public health nurses' home-visit skills during their first year of practice. DESIGN AND SAMPLE: A one-year longitudinal observational study was conducted with 80 novice public health nurses. The participants were recruited from the four Japanese prefectures and their affiliated municipalities selected by using stratified sampling. MEASUREMENTS: Self-administered questionnaires were distributed three times between April 2016 and March 2017, asking about home-visit experiences, understandings of the significance of home-visit, and the levels of home-visit skills. Data were analyzed with paired t-tests and Wilcoxon signed-rank tests. RESULTS: All of twenty skills' self-ratings significantly increased between the fourth and eighth months; five skills continually increased between the eighth and twelfth months. Both of Physical Assessment and Relationship Development Skills improved to the "Can do, almost by myself" level at the twelfth month; other skills remained at the "Can do, with a little advice" level. CONCLUSION: Although novice public health nurses improved to conducted home-visits independently, they didn't perceive themselves as being able to do so in their first year. Therefore, through preparing guideline, organizations should provide intentional support to assist their skill acquisition during their first, and even in their second year.

Leukotriene A4 hydrolase deficiency protects mice from diet-induced obesity by increasing energy expenditure through neuroendocrine axis.

Obesity is a health problem worldwide, and brown adipose tissue (BAT) is important for energy expenditure. Here, we explored the role of leukotriene A4 hydrolase (LTA4 H), a key enzyme in the synthesis of the lipid mediator leukotriene B4 (LTB4 ), in diet-induced obesity. LTA4 H-deficient (LTA4 H-KO) mice fed a high-fat diet (HFD) showed a lean phenotype, and bone-marrow transplantation studies revealed that LTA4 H-deficiency in non-hematopoietic cells was responsible for this lean phenotype. LTA4 H-KO mice exhibited greater energy expenditure, but similar food intake and fecal energy loss. LTA4 H-KO BAT showed higher expression of thermogenesis-related genes. In addition, the plasma thyroid-stimulating hormone and thyroid hormone concentrations, as well as HFD-induced catecholamine secretion, were higher in LTA4 H-KO mice. In contrast, LTB4 receptor (BLT1)-deficient mice did not show a lean phenotype, implying that the phenotype of LTA4 H-KO mice is independent of the LTB4 /BLT1 axis. These results indicate that LTA4 H mediates the diet-induced obesity by reducing catecholamine and thyroid hormone secretion.

Identification, signaling, and functions of LTB4 receptors.

Leukotriene B4 (LTB4), a lipid mediator produced from arachidonic acid, is a chemoattractant for inflammatory leukocytes. We identified two receptors for LTB4, the high-affinity receptor BLT1 and the low-affinity receptor BLT2. BLT1 is expressed in various subsets of leukocytes, and analyses of BLT1-deficient mice revealed that the LTB4/BLT1 axis enhances leukocyte recruitment to infected sites, and is involved in the elimination of pathogens. Hyperactivation of the LTB4/BLT1 axis induces acute and chronic inflammation, resulting in various inflammatory diseases. BLT2 was originally identified as a low-affinity receptor for LTB4, and we later identified 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (12-HHT) as a high-affinity ligand for BLT2. BLT2 is highly expressed in epithelial cells in various tissues including intestine and skin. Large quantities of 12-HHT are produced by activated platelets during skin injury, and activation of BLT2 on epidermal keratinocytes accelerates skin wound healing by enhancing cell migration. BLT2 signaling also enhances cell-cell junctions, protectes against transepidermal water loss, and preventes entry of environmental substances into the body.

[Gaining consensus on key terms in community health activities: A national survey using the Delphi method].

Objectives　The purpose of this study was to define the terminology used in community health activities by systematically establishing agreement among public health nurses and related professionals, to aid them in effectively cooperating with other professionals.Methods　We extracted the major terms described in "Guidelines for Public Health Nurses' Activities in the Region," which was issued by the Director of Health Bureau, Ministry of Health, Labor and Welfare; and conducted 2 iterations on the proposed definition of the terms using the Delphi method. The survey targeted 800 stakeholders from the following 4 professions: public health nurses in managerial positions in local governments, clerical staff in local governments, public health nursing professors at educational institutions, and social welfare council staff. Respondents indicated their degree of agreement with the definition of the terms using a 4-point Likert scale. We tallied the answers indicating "I agree" or "Mostly agree." A 70% term agreement was considered "conformed." A free text box for each proposed definition was also available so that respondents could share their opinions and offer alternatives.Results　The first survey received 231 responses. The conformity of their definitions ranged from 83.9%-96.9% (mean=91.5%). Although all definitions exceeded the consensus level, we examined their adequacy with reference to the definitions and opinions from the free text. Various opinions were obtained from those engaged in each occupation. The second round included 117 consenting stakeholders from the first survey, yielding 90 responses. Their degree of conformity ranged from 86.7%-98.9% (mean=94.6%). Therefore, we determined the definitions had reached a robust agreement. We revised the definitions of some terms with reference to the opinions from the free texts and finalized the definitions. Finally, we defined 23 terms-such as region, community diagnosis, and policy.Conclusion　All terms had a conformity of more than 85%, thus bringing into alignment those community health terms that had varying interpretations among related occupations earlier. By including the opinions of those from the related occupations who collaborate with public health nurses, the definitions of these terms could be established and shared. These definitions can be used in public health nursing practice, education, service, and research-among those in involved these related occupations.

Leukotriene B4 Receptor Type 2 Accelerates the Healing of Intestinal Lesions by Promoting Epithelial Cell Proliferation.

Leukotriene B4 receptor type 2 (BLT2) is a low-affinity leukotriene B4 receptor that is highly expressed in intestinal epithelial cells. Previous studies demonstrated the protective role of BLT2 in experimentally induced colitis. However, its role in intestinal lesion repair is not fully understood. We investigated the role of BLT2 in the healing of indomethacin-induced intestinal lesions in mice. There was no significant different between wild-type (WT) and BLT2-deficient (BLT2KO) mice in terms of the development of indomethacin-induced intestinal lesions. However, healing of these lesions was significantly impaired in BLT2KO mice compared with WT mice. In contrast, transgenic mice with intestinal epithelium-specific BLT2 overexpression presented with superior ileal lesion healing relative to WT mice. An immunohistochemical study showed that the number of Ki-67-proliferative cells was markedly increased during the healing of intestinal lesions in WT mice but significantly attenuated in BLT2KO mice. Exposure of cultured mouse intestinal epithelial cells to CAY10583, a BLT2 agonist, promoted wound healing and cell proliferation in a concentration-dependent manner. Nevertheless, these responses were abolished under serum-free conditions. The CAY10583-induced proliferative effect was also negated by Go6983, a protein kinase C (PKC) inhibitor, U-73122, a phospholipase C (PLC) inhibitor, LY255283, a BLT2 antagonist, and pertussis toxin that inhibits G protein-coupled receptor signaling via Gi/o proteins. Thus, BLT2 plays an important role in intestinal wound repair. Moreover, this effect is mediated by the promotion of epithelial cell proliferation via the Gi/o protein-dependent and PLC/PKC signaling pathways. The BLT2 agonists are potential therapeutic agents for the treatment of intestinal lesions. SIGNIFICANCE STATEMENT: The healing of indomethacin-induced Crohn's disease-like intestinal lesions was impaired in mice deficient in low-affinity leukotriene B4 receptor type 2 (BLT2). They presented with reduced epithelial cell proliferation during the healing. In contrast, healing was promoted in mice overexpressing intestinal epithelial BLT2. In cultured intestinal epithelial cells, the BLT2 agonist CAY10583 substantially accelerated wound repair by enhancing cell proliferation rather than migration. Thus, BLT2 plays an important role in the intestinal lesions via acceleration of epithelial cell proliferation.

Dietary ω-3 fatty acids alter the lipid mediator profile and alleviate allergic conjunctivitis without modulating Th2 immune responses.

Allergic conjunctivitis (AC) is one of the most common ocular surface diseases in the world. In AC, T helper type 2 (Th2) immune responses play central roles in orchestrating inflammatory responses. However, the roles of lipid mediators in the onset and progression of AC remain to be fully explored. Although previous reports have shown the beneficial effects of supplementation of ω-3 fatty acids in asthma or atopic dermatitis, the underlying molecular mechanisms are poorly understood. In this study, a diet rich in ω-3 fatty acids alleviated AC symptoms in both early and late phases without affecting Th2 immune responses, but rather by altering the lipid mediator profiles. The ω-3 fatty acids completely suppressed scratching behavior toward the eyes, an allergic reaction provoked by itch. Although total serum IgE levels and the expression levels of Th2 cytokines and chemokines in the conjunctiva were not altered by ω-3 fatty acids, eosinophil infiltration into the conjunctiva was dramatically suppressed. The levels of ω-6-derived proinflammatory lipid mediators, including those with chemoattractant properties for eosinophils, were markedly reduced in the conjunctivae of ω-3 diet-fed mice. Dietary ω-3 fatty acids can alleviate a variety of symptoms of AC by altering the lipid mediator profile.-Hirakata, T., Lee, H.-C., Ohba, M., Saeki, K., Okuno, T., Murakami, A., Matsuda, A., Yokomizo, T. Dietary ω-3 fatty acids alter the lipid mediator profile and alleviate allergic conjunctivitis without modulating Th2 immune responses.

Developing a comprehensive career development scale for public health nurses in Japan.

OBJECTIVES: This study aims to develop a scale that can comprehensively measure public health nurses'(PHNs) career development and to verify the validity and reliability of that scale. DESIGN AND SAMPLES: This study is a cross-sectional investigation. The participants were 1,009 PHNs working in a government facility in Japan. A total of 586 participants who had been working for more than 3 years as PHNs were analyzed. MEASUREMENTS: Anonymous self-response questionnaires included items on professional awareness, practical competency, and job satisfaction. We conducted exploratory factor analysis and confirmatory factor analysis to clarify the construct validity of the scale. The reliability was examined by the Cronbach's α coefficient. The validity was examined by an external reference relevant validity and the known-group method. RESULTS: The scale was structured with 32 items covering three factors: "community activities, policy, and management," "PHN identity," and "foundations as a PHN." The Cronbach's α coefficients were all greater than 0.9. In confirmatory factor analysis, the scale showed acceptable goodness of fit. CONCLUSIONS: The comprehensive career development scale for PHNs proved its reliability and validity. This scale may be useful to promote PHN identity and competency comprehensively in basic education and in-charge education.

Mothers' challenges with the parental control of 3-year-old children in Japan: A qualitative study.

BACKGROUND: Parents often struggle against the negativism of 3-year-old children in exerting parental control. Although most research on parental control is from western cultures, Japanese mothers may face unique challenges because of their sociocultural background. We therefore explored mothers' challenges in exerting parental control over 3-year-old children in Japan. METHODS: A descriptive qualitative approach was adopted. The participants were 10 Japanese mothers with children aged 3-4 years. We conducted individual semistructured interviews and analysed the data via qualitative content analysis. RESULTS: We found four categories and a structure of the challenges mothers faced in exerting parental control. Mothers' control attempts had four main intentions, each with a different subject (i.e., the children, themselves, the family, and society). Most challenges with parental control stemmed from mothers' "consciousness of parental responsibility." In confronting the child in their attempts to exert parental control, mothers perceived a "struggle with the child's negativism," which led them to recognize that they could not find a correct method of discipline. Furthermore, in the face of conflicting roles, mothers experienced "confusion in seeking the righteous answer as a parent." Some mothers began to feel that they were discovering their maternal role in their own way rather than finding a correct answer, which formed a "conviction that my own parenting is not incorrect." CONCLUSIONS: The challenges mothers faced in exerting parental control over their 3-year-old children served as a turning point for them to reflect on themselves as parents. However, due to societal monitoring of parents and mothers' multiple roles in the family, it can be difficult for mothers to fully overcome the challenges of parental control. It is necessary to foster social capital in the community to which the mother belongs and to create an atmosphere in which community members can watch over parents warmly.

[Risk perception of tsunamis and the willingness to evacuate of junior high school students living in coastal areas].

Objective　Students find it difficult to take the decision to evacuate from tsunamis. This study explores junior high school students' risk perception regarding tsunamis and their willingness to evacuate.Methods　The study surveyed 251 junior high school students from the 7th to 9th grades in Town B, located at the east coast of Prefecture A, Japan, using data from an anonymous questionnaire that was administered with parental consent. Demographic factors (school, grade, and gender), willingness to evacuate, risk perception, tsunami experiences, recognition of tsunami hazard households, and household preventive actions were evaluated. Pearson's chi-squared test and Fisher's exact test were used to analyze the relationship between risk perception and willingness to evacuate. The study was approved by the Ethics Committee of Hokkaido University.Results　Valid data were collected from 158 students (62.9%). Of these, 141 (89.2%) were enrolled in a school located in a coastal area. Male students accounted for 81 responses (51.3%) and female students for 77 (48.7%). As for willingness to evacuate, 147 (93.0%) responded that they would evacuate if they heard an evacuation order and 112 (70.9%) responded that they would evacuate if they experienced a persistent tremor. Regarding household altitude, 66 (41.8%) of the students live in houses located less than 15 meters above sea level. The results indicate that 125 (79.1%) of the students discuss tsunamis with their family members. The primary factor promoting the willingness to evacuate after hearing an evacuation order is living in a household under 15 meters in altitude (100% vs. 82.4%, P<0.001) and the major factors for willingness to evacuate after feeling a persistent tremor are living in a household under 15 meters in altitude (84.8% vs. 35.3%, P<0.001) and discussing tsunamis with their families (76.8% vs. 48.5%, P=0.001).Conclusion　The results suggest that to promote a willingness to evacuate immediately, recognizing the household's altitude is important, as it can give junior high school students a clearer sense of the danger of tsunamis. In addition, the study suggests that discussing the matter within families is a significant factor that promotes willingness to evacuate.

Leukotriene B4 receptor 2 regulates the proliferation, migration, and barrier integrity of bronchial epithelial cells.

The airway epithelium plays a crucial role in the pathogenesis of asthma. The functions of leukotriene B4 receptor 2 (BLT2) on the airway epithelial cells remains unknown. In our study, BLT2 expression in 16HBE bronchial epithelial cells were manipulated by transfection with BLT2 overexpression plasmid or BLT2 small interference RNA. 16HBE cells were then exposed to BLT2 antagonist (LY255283) or BLT2 agonist (12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid [12-HHT] or CAY10583). The results showed that BLT2 overexpression, 12-HHT stimulation, or CAY10583 treatment resulted in the enhanced proliferation and migration of 16HBE cells. In addition, BLT2 showed an inhibitory effect on epithelial permeability as illustrated by the measurement of transepithelial electrical resistance (TER) and epithelial permeability, and a promoting effect on the levels of tight junction proteins (occludin and claudin-4) and phosphorylated p38 as demonstrated by real-time PCR and Western blotting analyses. These results suggest BLT2 as a key determinant of airway epithelial barrier integrity. On the contrary, RNAi-mediated knockdown or LY255283 treatment had reversed effects on the proliferation, migration, and epithelial barrier integrity. Together, our findings suggest the critical roles of BLT2 on the functions of bronchial epithelial cells and that BLT2 agonists are potential therapeutic agents for asthma treatment.

Profiling of bioactive lipids in different dendritic cell subsets using an improved multiplex quantitative LC-MS/MS method.

Lipids are an energy source and key components of the cell membrane; however, they are also bioactive mediators of physiological and pathophysiological phenomena. Quantification of bioactive lipids is not easy because they have diverse chemical properties and are present in trace amounts. Here, we improved a multiplex method of quantifying bioactive lipids, thereby enabling measurement of 90 compounds simultaneously. We then used this system to quantify bioactive lipids produced by two subsets of dendritic cells (DCs): all-trans retinoic acid-treated DCs (RA-DCs) (a type of tolerogenic DC (tDC)) and conventional DCs (cDCs). We found that cDCs produced inflammatory lipid mediators such as leukotrienes, whereas RA-DCs produced anti-inflammatory lipid mediators such as prostaglandin I2. Consistent with this, cDCs expressed larger amounts of mRNA encoding 5-lipoxygenase and LTA4 hydrolase (both responsible for leukotriene biosynthesis) and RA-DCs produced larger amounts of mRNA encoding prostaglandin I2 synthase. Taken together, the results suggest that the method is useful for clarifying the roles of bioactive lipids during immune responses.

Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type-Specific Manner.

The clathrin-dependent endocytic pathway is crucial for endosomal TLR3- and TLR4-mediated Toll-IL-1R domain-containing adaptor molecule-1 (TICAM-1) signaling. TLR4 uses a different signaling platform, plasma membrane and endosomes, for activation of TIRAP-MyD88 and TICAM-2-TICAM-1, respectively. LPS-induced endocytosis of TLR4 is mandatory for TICAM-1-mediated signaling including IFN-ß production. Several molecules/mechanisms such as CD14, clathrin, and phosphatidylinositol metabolism have been reported to act as inducers of TLR4 translocation. However, the molecular mechanism of spatiotemporal regulation of TLR4 signaling remains unresolved. We have previously shown that Raftlin is essential for clathrin-dependent endocytosis of TLR3 ligand in human epithelial cells and myeloid dendritic cells (DCs). In this article, we demonstrate that Raftlin also mediated LPS-induced TLR4 internalization and TICAM-1 signaling in human monocyte-derived DCs and macrophages (Mo-Mϕs). When Raftlin was knocked down, LPS-induced TLR4-mediated IFN-ß promoter activation, but not NF-κB activation, was decreased in HEK293 cells overexpressing TLR4/MD-2 or TLR4/MD-2/CD14. LPS-induced IFN-ß production by monocyte-derived DCs and Mo-Mϕs was significantly decreased by knockdown of Raftlin. Upon LPS stimulation, Raftlin moved from the cytoplasm to the plasma membrane in Mo-Mϕs, where it colocalized with TLR4. Raftlin associated with clathrin-associated adaptor protein-2 in resting cells and transiently bound to TLR4 and clathrin at the cell surface in response to LPS. Thus, Raftlin appears to modulate cargo selection as an accessary protein of clathrin-associated adaptor protein-2 in clathrin-mediated endocytosis of TLR3/4 ligands.

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues.

Leukotriene B4 receptor type 2 (BLT2) enhances skin barrier function by regulating tight junction proteins.

GPCRs are involved in numerous physiologic functions and are important drug targets. Although the epithelial barrier is important for protection from invading pathogens, the correlation between GPCRs and epithelial barrier function remains unknown. Leukotriene B4 (LTB4) receptor type 2 (BLT2), mainly expressed in epithelial cells, is a GPCR for 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT) and LTB4. In our study, BLT2 localized at the lateral membrane in BLT2-overexpressing Madin-Darby canine kidney (MDCK) II cells and in the small intestine of BLT2-transgenic mice. BLT2-deficient mice exhibited higher transepidermal water loss and were more sensitive to epicutaneous sensitization. MDCK-BLT2 cells recovered transepithelial electrical resistance (TER) after a calcium switch faster than did MDCK-Mock cells, and 12-HHT stimulation accelerated TER recovery only in MDCK-BLT2 cells. Quantitative PCR and immunoblot analyses revealed that the 12-HHT/BLT2 axis up-regulated claudin-4 (CLDN4) expression in MDCK-BLT2 cells and human primary keratinocytes, and CLDN4 knockdown abolished 12-HHT-dependent TER recovery. Acceleration of TER recovery and induction of CLDN4 expression by 12-HHT stimulation were abolished by inhibition of Gαi protein or p38 MAPK. These results show that 12-HHT/BLT2 enhances epithelial barrier function by increasing CLDN4 expression via the Gαi protein-p38 MAPK pathway.