Kyoko Futamura

IL-33 Mediates Inflammatory Responses in Human Lung Tissue Cells

IL-33 is a member of the IL-1 family and mediates its biological effects via the ST2 receptor, which is selectively expressed on Th2 cells and mast cells. Although polymorphic variation in ST2 is strongly associated with asthma, it is currently unclear whether IL-33 acts directly on lung tissue cells at sites of airway remodeling. Therefore, we aimed to identify the IL-33–responsive cells among primary human lung tissue cells. ST2 mRNA was expressed in both endothelial and epithelial cells but not in fibroblasts or smooth muscle cells. Correspondingly, IL-33 promoted IL-8 production by both endothelial and epithelial cells but not by fibroblasts or smooth muscle cells. Transfection of ST2 small interference RNA into both endothelial and epithelial cells significantly reduced the IL-33–dependent upregulation of IL-8, suggesting that IL-33–mediated responses in these cells occur via the ST2 receptor. Importantly, Th2 cytokines, such as IL-4, further enhanced ST2 expression and function in both endothelial and epithelial cells. The IL-33–mediated production of IL-8 by epithelial cells was almost completely suppressed by corticosteroid treatment. In contrast, the effect of corticosteroid treatment on the IL-33–mediated responses of endothelial cells was only partial. IL-33 induced activation of both ERK and p38 MAPK in endothelial cells but only ERK in epithelial cells. p38 MAPK was required for the IL-33–mediated responses of endothelial cells, whereas ERK was required for IL-33–mediated IL-8 production by epithelial cells. Taken together, these findings suggest that IL-33–mediated inflammatory responses of lung tissue cells may be involved in the chronic allergic inflammation of the asthmatic airway.

The Unfolded Protein Response Is Activated in Differentiating Epidermal Keratinocytes

The unfolded protein response (UPR), which is induced by stress to the endoplasmic reticulum (ER), is involved in the functional alteration of certain cells, such as the differentiation of B cells to plasma cells. The aim of this study is to determine whether the UPR is activated during epidermal keratinocyte (KC) differentiation. Here, we show that the expression of the UPR-induced proteins Bip/GRP78 and HRD1 was increased in cells in the supra-basal layers of normal human epidermis that contain KCs undergoing differentiation as well as in skin-equivalent cultured KCs. However, Bip/GRP78 and HRD1 were poorly expressed in proliferating KCs in squamous cell carcinoma and psoriasis vulgaris tissues. The epidermal growth factor receptor tyrosine kinase inhibitor, PD153035, which induces KC differentiation, upregulated UPR-induced marker mRNAs and proteins. Furthermore, microarray analyses and quantitative PCR revealed that ER stress-inducing reagents, tunicamycin (TU), thapsigargin, and brefeldin A, altered the expression of genes essential for human epidermal KC differentiation, including C/EBPbeta, KLF4, and ABCA12 in vitro. However, ABCA12 and KLF4 mRNA did not increase with TU treatment after siRNA-mediated knockdown of XBP-1. Taken together, our findings strongly suggest that the UPR is activated during normal epidermal KC differentiation and induces C/EBPbeta, KLF4, and ABCA12 mRNAs.

β2-Adrenoceptor Agonists Enhance Cytokine-Induced Release of Thymic Stromal Lymphopoietin by Lung Tissue Cells

Background: Whileβ2-adrenoceptor agonists (β2-agonists) are widely used as bronchodilators in the treatment of asthma, there has been increasing concern that regular use of β2-agonists may adversely affect the control of asthma. However, the molecular mechanisms of such undesirable effects of β2-agonists are not fully understood. In this study, we examined the effects of β2-agonists on cytokine-induced production of thymic stromal lymphopoietin (TSLP), an indispensable cytokine in the development of allergic diseases, by lung tissue cells. Methods: Normal human bronchial epithelial cells (NHBE), smooth muscle cells (BSMC) and fibroblasts (NHLF) were stimulated with the IL-4 and TNF-α cytokines, alone and in combination, and their production of TSLP was examined by ELISA. The effects of β2-agonists (salmeterol, formoterol, salbutamol), intracellular cyclic adenosine monophosphate (cAMP)-elevating agents (8-bromo-cAMP, dibutyryl cAMP, forskolin) and a corticosteroid (fluticasone) on the cytokine-induced TSLP production were examined. Results: The following results were observed in all three types of lung tissue cells tested (that is, NHBE, BSMC and NHLF). Costimulation with IL-4 and TNF-α significantly induced TSLP production, and β2-agonists further enhanced it via upregulation of intracellular cAMP. However, addition of a corticosteroid to the cytokines and β2-agonist resulted in a marked decrease in TSLP production. Conclusions: β2-Agonists significantly enhanced the cytokine-induced TSLP production by primary human lung tissue cells. This may be partly responsible for the undesirable clinical effects of continuous β2-agonist monotherapy, and combination therapy with a corticosteroid might effectively inhibit TSLP-mediated allergic inflammation.

Cell type‐dependent effects of corticosteroid on periostin production by primary human tissue cells

Overproduction of periostin, an IL‐13‐inducible matricellular protein, despite corticosteroid treatment is thought to be involved in the chronicity of allergic inflammation seen in corticosteroid‐refractory tissue fibrosis. Therefore, we hypothesized that some tissue cells must produce periostin in a corticosteroid‐insensitive manner. Here, we show that IL‐4 and IL‐13 each induced comparable levels of periostin production by primary normal human fibroblasts and microvascular endothelial cells derived from lung and skin. Dexamethasone, a corticosteroid, completely inhibited IL‐4/13‐induced, but did not affect TGF‐β‐induced, periostin production by fibroblasts. In contrast, dexamethasone synergistically enhanced IL‐4/13‐induced periostin production by microvascular endothelial cells. TGF‐β did not induce periostin production by microvascular endothelial cells. Our novel findings suggest that IL‐4/13‐induced microvascular endothelium‐derived and/or TGF‐β‐induced fibroblast‐derived periostin might play a pivotal role in corticosteroid‐refractory tissue fibrosis, leading to chronic allergic inflammation in the lung and/or skin.

Overexpression of LEDGF/DFS70 induces IL-6 via p38 activation in HaCaT cells, similar to that seen in the psoriatic condition.

Lens epithelium-derived growth factor (LEDGF)/dense fine speckles 70  kDa protein (DFS70) is a transcription cofactor that enhances growth and is overexpressed in various cancers. In the epidermis, LEDGF/DFS70 localizes to the nucleus of keratinocytes (KCs) in the basal layers and to the cytoplasm of cells in the upper layers. However, the biological and pathological relevance of LEDGF/DFS70 in the epidermis is virtually unknown. Compared with normal epidermis, we detected strong nuclear staining of LEDGF/DFS70 in both the spinous and basal layers of the epidermis of psoriatic skin. To investigate the roles of LEDGF/DFS70 in the epidermis of psoriatic skin, we generated HaCaT cells that constitutively express enhanced green fluorescence protein (EGFP)-LEDGF (EGFP-LEDGF-HaCaT) or EGFP alone (EGFP-HaCaT) as a control. EGFP-LEDGF-HaCaT cells had increased expression of IL-6, which was attenuated by LEDGF-specific RNA interference and the p38-specific inhibitors SB-239063 and SB-203580. Furthermore, EGFP-LEDGF-HaCaT cells had increased expression of S100A7 and S100A9 and decreased expression of filaggrin. These findings are compatible with the expression pattern in psoriatic tissues. Taken together, these results strongly suggest that ectopic expression of LEDGF/DFS70 in KCs could be involved in the pathology of psoriasis vulgaris.

Infantile Eczema at One Month of Age Is Associated with Cord Blood Eosinophilia and Subsequent Development of Atopic Dermatitis and Wheezing Illness until Two Years of Age

Background: Physiological and pathological skin eruptions are commonly encountered in neonates in our clinical practice. However, the types of skin eruptions that are associated with the subsequent development of atopic dermatitis and the mechanisms of these associations remain uncertain. Methods: A total of 105 newborn babies with normal delivery were enrolled in this prospective cohort study. The cord blood eosinophil count was measured and the neonates were examined at 1 month of age and followed until 8 years of age. Results: At 1 month of age, infantile eczema, seborrheic dermatitis, intertrigo and diaper dermatitis were diagnosed in a total of 29, 7, 14 and 24 neonates, respectively. No association was found among the prevalences of these eruptions. Neonates with infantile eczema had a significantly higher number and ratio of eosinophils in the cord blood (eosinophil count: 670.8 ± 67.8 vs. 349.0 ± 30.3/µl, p < 0.0001; eosinophil ratio: 5.12 ± 0.53 vs. 2.61 ± 0.22%, p < 0.0001, for the presence and the absence of infantile eczema, respectively). In contrast, no such tendency was found for any other skin eruptions. In neonates with infantile eczema at 1 month of age, the diagnosis of atopic dermatitis had been made significantly earlier and the prevalence of wheezing illness was significantly higher than in those without infantile eczema until 2 years of age. Conclusion: Infantile eczema, but not other skin eruptions, precedes the development of atopic dermatitis and wheezing illness during early infancy, presumably because of the activation of eosinophils before birth.

Recent advances in understanding the roles of vascular endothelial cells in allergic inflammation

Allergic disorders commonly involve both chronic tissue inflammation and remodeling caused by immunological reactions to various antigens on tissue surfaces. Due to their anatomical location, vascular endothelial cells are the final responders to interact with various exogenous factors that come into contact with the epithelial surface, such as pathogen-associated molecular patterns (PAMPs) and antigens. Recent studies have shed light on the important roles of endothelial cells in the development and exacerbation of allergic disorders. For instance, endothelial cells have the greatest potential to produce several key molecules that are deeply involved in allergic inflammation, such as periostin and thymus and activation-regulated chemokine (TARC/CCL17). Additionally, endothelial cells were recently shown to be important functional targets for IL-33--an essential regulator of allergic inflammation. Notably, almost all endothelial cell responses and functions involved in allergic inflammation are not suppressed by corticosteroids. These corticosteroid-refractory endothelial cell responses and functions include TNF-α-associated angiogenesis, leukocyte adhesion, IL-33-mediated responses and periostin and TARC production. Therefore, these unique responses and functions of endothelial cells may be critically involved in the pathogenesis of various allergic disorders, especially their refractory processes. Here, we review recent studies, including ours, which have elucidated previously unknown pathophysiological roles of vascular endothelial cells in allergic inflammation and discuss the possibility of endothelium-targeted therapy for allergic disorders.

Expression of thymus and activation-regulated chemokine (TARC) by human dermal cells, but not epidermal keratinocytes

BACKGROUND Serum levels of thymus and activation-regulated chemokine (TARC/CCL17) have served as a reliable biomarker of disease progression of atopic dermatitis (AD). However, it remains to be scientifically explained why serum TARC levels correlate well with the degree of AD progression. OBJECTIVE We hypothesized that dermal cells, but not epidermal keratinocytes, are major cellular sources of TARC and thus responsible for subclinical skin inflammation. This study aimed to identify the skin cells that can produce TARC protein. METHODS Primary normal human epidermal keratinocytes (NHEK), dermal microvascular endothelial cells (HMVEC-dBl) and dermal fibroblasts (NHDF) were stimulated with TNF-α and IL-4, alone and in combination. TARC mRNA and protein levels were quantified by qPCR and ELISA, respectively. We also investigated the effects of such immunosuppressants as a corticosteroid (dexamethasone) and tacrolimus (FK506) on TARC production, and used various signaling inhibitors to evaluate the signaling pathways involved in TARC expression. RESULTS Although neither TNF-α nor IL-4 alone induced TARC production by any of the tested cell types, together they induced expression of TARC mRNA and appreciable amounts of TARC protein by HMVEC-dBl and NHDF, but not by NHEK. TARC production by those dermal cells was not inhibited by dexamethasone or FK506. TARC production by HMVEC-dBl was completely inhibited by NF-κB and p38 MAPK inhibitors, but not by an ERK inhibitor. CONCLUSION Dermal cells, but not epidermal keratinocytes, may be important cellular sources of TARC in AD skin. Therefore, even if epidermal eczematous lesions seem to be improved, complete inhibition of inflammation in the dermis is thought to be particularly important for suppressing both the TARC blood level and progression of AD. However, immunosuppressants did not directly inhibit TARC production by the dermal cells. Anti-inflammatory therapy may decrease TARC blood levels in AD patients indirectly, via its inhibitory effects on TNF-α- and/or IL-4-producing cells in the dermis.

Silica and Double-Stranded RNA Synergistically Induce Bronchial Epithelial Apoptosis and Airway Inflammation

Silica crystals (silica), which are the main mineral component of volcanic ash and desert dust, can activate the caspase-1-activating inflammasome in phagocytic cells to secrete IL-1β. Although inhalation of silica-containing dust is known to exacerbate chronic respiratory diseases, probably through inflammasome activation, its direct effects on bronchial epithelial cells remain unclear. Here, we show that silica and double-stranded RNA (dsRNA) synergistically induces caspase-9-dependent apoptosis, but not inflammasome activation, of bronchial epithelial cells. Intranasal administration of silica and dsRNA to mice synergistically enhanced neutrophil infiltration in the airway without IL-1β release in the bronchoalveolar lavage fluid. Histopathological analysis revealed that silica or dsRNA alone induced slight airway inflammation, whereas combined administration significantly enhanced airway inflammation and epithelial damage. These novel findings suggest that inhalation of silica-containing dust may cause inflammasome-independent airway inflammation, possibly by damaging the epithelial barrier, especially at the time of viral infection. These responses may also be involved in acute lung injury caused by inhaled silica-containing dust.

Gene expression profiling of acute type A aortic dissection combined with in vitro assessment

OBJECTIVES The mechanisms underlying aortic dissection remain to be fully elucidated. We aimed to identify key molecules driving dissection through gene expression profiling achieved by microarray analysis and subsequent in vitro experiments using human aortic endothelial cells (HAECs) and aortic vascular smooth muscle cells (AoSMCs). METHODS Total RNA, including microRNA (miRNA), was isolated from the intima‐media layer of dissected ascending aorta obtained intraoperatively from acute type A aortic dissection (ATAAD) patients without familial thoracic aortic disease (n = 8) and that of non‐dissected ascending aorta obtained from transplant donors (n = 9). Gene expression profiling was performed with mRNA and miRNA microarrays, and results were confirmed by quantitative polymerase chain reaction (qPCR). Target genes and miRNA were identified by gene ontology analysis and a literature search. To reproduce the in silico results, HAECs and AoSMCs were stimulated in vitro by upstream cytokines, and expression of target genes was assessed by qPCR. RESULTS Microarray analysis revealed 1536 genes (3.6%, 1536/42 545 probes) and 41 miRNAs (3.0%, 41/1368 probes) that were differentially expressed in the ATAAD group (versus donor group). The top 15 related pathways included regulation of inflammatory response, growth factor activity and extracellular matrix. Gene ontology analysis identified JAK2 (regulation of inflammatory response), PDGFA, TGFB1, VEGFA (growth factor activity) and TIMP3, TIMP4, SERPINE1 (extracellular matrix) as the target genes and miR‐21‐5p, a TIMP3 repressor, as target miRNA that interacts with the target genes. Validation qPCR confirmed the altered expression of all 7 target genes and miR‐21‐5p in dissected aorta specimens (all genes, P < 0.05). Ingenuity pathway analysis showed TNF‐&agr; and TGF‐&bgr; to be upstream cytokines for the target genes. In vitro experiments showed these cytokines inhibit TIMP3 expression (P < 0.05) and enhance VEGFA expression (P < 0.01) in AoSMCs but not HAECs. miR‐21‐5p expression increases in AoSMCs under TNF‐&agr; and TGF‐&bgr; stimulation (fold change: 1.36; P = 0.011). CONCLUSIONS Results of our novel approach, integrating in vitro assessment into gene expression profiling, implicated chronic inflammation characterized by MMP‐TIMP dysregulation, increased VEGFA expression, and TGF‐&bgr; signalling in the development of dissection. Further investigation may reveal novel diagnostic biomarkers and uncover the mechanism(s) underlying ATAAD.