Gelatin promotes murine fibrosarcoma L929 cell detachment and protects the cells from TNFα-induced cytotoxicity

Purpose: Gelatin has been considered to exist as intermediate substance of collagen catabolism in tissue remodeling or under inflammatory conditions. We have initiated the study on possible biological functions of gelatin that can exist temporally and locally under the conditions of remodeling and inflammation Materials and methods: To this purpose, we investigated cell proliferation and survival on gelatin-coated dishes and the response to tumor necrosis factor α (TNFα)–induced cytotoxicity in L929 cells. Autophagy level, ATP level, and ROS generation are examined. Results: L929 cells detached from the gelatin-coated dishes and formed multicellular aggregates. TNFα-induced cytotoxicity in L929 cells was inhibited by gelatin-coating culture. The cells on gelatin-coated dishes showed reduced cellular ATP levels and increased adenosine 5′-monophosphate (AMP)–activated protein kinase (AMPK) phosphorylation, leading to increased ROS generation and autophagy. Conclusion: This study showed that gelatin-coated culture protected L929 cells from TNFα-induced cytotoxicity and suggested for a possible pathophysiological function of gelatin in regulating cellular functions.     

IKK-2 is required for TNF-α-induced invasion and proliferation of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) can contribute to tissue repair by actively migrating to sites of tissue injury. However, the cellular and molecular mechanisms of MSC recruitment are largely unknown. The nuclear factor (NF)-κB pathway plays a pivotal role in regulating genes that influence cell migration, cell differentiation, inflammation, and proliferation. One of the major cytokines released at sites of injury is tumor necrosis factor-α (TNF-α), which is known to be a key regulator of the NF-κB pathway. Therefore, we hypothesized that TNF-α may lead to MSC invasion and proliferation by activation of the NF-κB pathway. TNF-receptor 1 and 2, NF-κB (p65), and IκB kinase 2 (IKK-2) are expressed in human MSCs (hMSCs). Stimulation of hMSCs with TNF-α caused a p65 translocation from the cytoplasm to nucleoplasm but did not change the expression profile of MSC markers. TNF-α strongly augmented the migration of hMSCs through the human extracellular matrix. Using lentiviral gene transfer, overexpressing a dominant-negative mutant of IKK-2 (dn-IKK-2) significantly blocked this effect. NF-κB target genes associated with migration (vascular cell adhesion molecule-1, CD44, and matrix metalloproteinase 9) were upregulated by TNF-α stimulation and blocked by dn-IKK-2. Moreover, using the bromodeoxyuridine assay, we showed that the inhibition of the NF-κB pathway caused a significant reduction in the basal proliferation rate. TNF-α stimulated the proliferation of hMSCs, whereas overexpression of dn-IKK-2 significantly blocked this effect. TNF-α led to the upregulated expression of the proliferation-associated gene cyclin D1. In conclusion, we demonstrated that the NF-κB pathway components, p65 and IKK-2, are expressed in hMSCs. Our data provide evidence that this signal transduction pathway is implicated in TNF-α-mediated invasion and proliferation of hMSCs. Therefore, hMSC recruitment to sites of tissue injury may, at least in part, be regulated by the NF-κB signal transduction pathway.     

Simvastatin attenuates TNF-α-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells

Objective and design  

Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that induces apoptosis in a number of cell systems, including osteoblasts. Simvastatin is a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor known as a stimulator for bone formation via promoting osteoblast differentiation and mineralization. This study was designed to examine the role of simvastatin on TNF-α-induced growth inhibition and apoptosis in murine osteoblastic MC3T3-E1 cells.     

TNF-α-converting enzyme/a disintegrin and metalloprotease-17 mediates mechanotransduction in murine tracheal epithelial cells

Bronchoconstriction applies compressive stress to airway epithelial cells. We show that the application of compressive stress to cultured murine tracheal epithelial cells elicits the increased phosphorylation of extracellular signal-regulated kinase (ERK) and Akt through an epidermal growth factor receptor (EGFR)-dependent process, consistent with previous observations of the bronchoconstriction-induced activation of EGFR in both human and murine airways. Mechanotransduction requires metalloprotease activity, indicating a pivotal role for proteolytic EGF-family ligand shedding. However, cells derived from mice with targeted deletions of the EGFR ligands Tgfα and Hb-egf showed only modest decreases in responses, even when combined with neutralizing antibodies to the EGFR ligands epiregulin and amphiregulin, suggesting redundant or compensatory roles for individual EGF family members in mechanotransduction. In contrast, cells harvested from mice with a conditional deletion of the gene encoding the TNF-α-converting enzyme (TACE/ADAM17), a sheddase for multiple EGF-family proligands, displayed a near-complete attenuation of ERK and Akt phosphorylation responses and compressive stress-induced gene regulation. Our data provide strong evidence that TACE plays a critical central role in the transduction of compressive stress.     

Acetylcholine-evoked calcium mobilization and ion channel activation in human labial gland acinar cells from patients with primary Sjögren's syndrome

Recent evidence has indicated that the salivary gland dysfunction associated with Sjögren's syndrome (SjS) is not necessarily due to immune‐mediated destruction of acinar tissue. SjS sufferers may possess substantial reserves of acinar tissue but nevertheless be incapable of maintaining salivary flow rates in the normal range. We have investigated the ability of isolated labial gland acinar cells from SjS patients to fluid secrete by measuring agonist‐evoked changes in intracellular Ca²⁺ ([Ca²⁺]_i) using fura‐2 microfluorimetry and activation of K⁺ and Cl^? channels using the patch‐clamp whole cell technique. We can confirm that stimulation with a super‐maximal dose of acetylcholine (ACh) increased [Ca²⁺]_i equally in both control acinar cells and those derived from SjS patients. However, at submaximal concentrations, the dose–response curve for ACh was shifted to the right by approximately one order of magnitude in acinar cells from SjS patients compared to control acinar cells. Patch‐clamp measurements consistent with the presence of Ca²⁺‐activated K⁺ and Cl^? conductances were obtained from both control acinar cells and those obtained from SjS patients. Dose‐dependent activation of the ion channels by acetylcholine was also right‐shifted in acinar cells from SjS patients compared to control cells. Our data show that labial gland acinar cells from SjS patients were capable of responding to agonist stimulation by mobilizing [Ca²⁺]_i and activating K⁺ and Cl^? channels consistent with the requirements of fluid secretion. However, the persistent loss of sensitivity to ACh observed in from SjS patients may account for the lack of saliva production observed in these patients in vivo.     

Vasoactive intestinal peptide/vasoactive intestinal peptide receptor relative expression in salivary glands as one endogenous modulator of acinar cell apoptosis in a murine model of Sjögren's syndrome

Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by a progressive oral and ocular dryness that correlates poorly with the autoimmune damage of the glands. It has been proposed that a loss of homeostatic equilibrium in the glands is partly responsible for salivary dysfunction with acinar cells involved actively in the pathogenesis of SS. The non‐obese diabetic (NOD) mouse model of Sjögren's syndrome develops secretory dysfunction and early loss of glandular homeostatic mechanisms, with mild infiltration of the glands. Based on the vasodilator, prosecretory and trophic effects of the vasoactive intestinal peptide (VIP) on acini as well as its anti‐inflammatory properties we hypothesized that the local expression of VIP/vasoactive intestinal peptide receptor (VPAC) system in salivary glands could have a role in acinar cell apoptosis and macrophage function thus influencing gland homeostasis. Here we show a progressive decline of VIP expression in submandibular glands of NOD mice with no changes in VPAC receptor expression compared with normal mice. The deep loss of endogenous VIP was associated with a loss of acinar cells through apoptotic mechanisms that could be induced further by tumour necrosis factor (TNF)‐α and reversed by VIP through a cyclic adenosine‐5′‐monophosphate (cAMP)/protein kinase A (PKA)‐mediated pathway. The clearance of apoptotic acinar cells by macrophages was impaired for NOD macrophages but a shift from inflammatory to regulatory phenotype was induced in macrophages during phagocytosis of apoptotic acinar cells. These results support that the decline in endogenous VIP/VPAC local levels might influence the survival/apoptosis intracellular set point in NOD acinar cells and their clearance, thus contributing to gland homeostasis loss.     

Non-proliferating plasma cells detected in the salivary glands and bone marrow of autoimmune NOD.B10.H2b mice,a model for primary Sjögren’s syndrome

Autoantibody secreting plasma cells (PCs) are essential contributors in the development of autoimmune conditions such as primary Sjögren’s syndrome (pSS). Particularly, the long-lived PC subset residing in the bone marrow has shown to continuously produce autoantibodies, whilst remaining unaffected by immunosuppressive treatment. We have previously shown accumulation of potentially long-lived PCs in chronically inflamed salivary glands of pSS patients. In this study, we aimed to characterise the PC compartment in the salivary glands (the target organ for pSS) and bone marrow before the onset of the murine pSS like disease versus advanced diseases progression. Bromodeoxyuridine (BrdU) was incorporated to distinguish the long-lived PCs. Double immunohistochemical staining and immunofluorescence were then conducted on submandibular gland and bone marrow sections from 8- and 40-week-old mice to identify BrdU and CD138. BrdU⁺ cells were detected in the submandibular glands of 8-week-old mice, and observed within all focal infiltrates by 40 weeks of age. Most CD138⁺ PCs were however BrdU^? and located predominantly on the periphery of these infiltrates. This observation was verified through immunofluorescence. A comparable staining pattern was observed in the bone marrow of 8- and 40-week-old NOD.B10.H2b mice, where some of the CD138⁺ cells also expressed BrdU. Interestingly, megakaryocytes in the bone marrow of NOD.B10.H2b mice were detected in close proximity to CD138⁺ cells, illustrating a possible presence of PC survival niches. Our results demonstrate the presence and accumulation of potentially long-lived PCs in NOD.B10.H2b mice as the disease advances.     

Isorhamnetin attenuates TNF-α-induced inflammation,proliferation, and migration in human bronchial epithelial cells via MAPK and NF-κB pathways

Isorhamnetin has distinct anti-inflammatory activity and inhibits cell proliferation and migration. These effects are also involved in the pathogenesis of asthma. However, the effect of isorhamnetin on bronchial epithelial cells in patients with asthma has not been examined. Cells of human bronchial epithelial cell line BEAS-2B were cultured with isorhamnetin and tumor necrosis factor (TNF)-α. The effects of isorhamnetin on BEAS-2B cell viability were assessed using CCK8 assay. The EdU (5-ethynyl-2′-deoxyuridine) cell proliferation assay was performed to assess cell proliferation. BEAS-2B cell migration was measured using Transwell and wound healing assays. Real-time PCR and enzyme-linked immunosorbent assay were conducted to measure the expression of pro-inflammatory cytokines. Protein expression levels were determined by western blotting. Immunofluorescence was used to detect nuclear translocation of nuclear factor kappa B (NF-κB). We found that isorhamnetin at 20 and 40 μM reduced the proliferation of BEAS-2B cells induced by TNF-α. Isorhamnetin significantly decreased the expression of interleukin (IL)-1β, IL-6, IL-8, and C-X-C motif chemokine ligand 10 in BEAS-2B cells induced by TNF-α. Additionally, 10 μM isorhamnetin effectively reduced cell migration induced by TNF-α. Treatment with isorhamnetin inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) and NF-κB pathways induced by TNF-α. In summary, isorhamnetin inhibited the inflammation, proliferation, and migration of BEAS-2B cells by regulating the MAPK and NF-κB signaling pathways and is a drug candidate for asthma.     

Expression of aquaporin-5 in and fluid secretion from immortalized human salivary gland ductal cells by treatment with 5-aza-2'-deoxycytidine: a possibility for improvement of xerostomia in patients with Sjögren's syndrome

The aim of the present study was to investigate the possibility that ductal cells, which preferentially survive and/or proliferate in Sj?gren's syndrome (SS) salivary glands of patients with SS, could acquire the functional expression of membrane water channel aquaporin-5 (AQP5). Thus, in this study, we demonstrate that an immortalized normal human salivary gland ductal cell (NS-SV-DC) line, lacking the expression of AQP5, acquires AQP5 gene expression in response to treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA demethylating agent. Confocal microscopic analysis revealed the localization of AQP5 expression mainly at the apical and lateral sides of the plasma membrane. The expressed AQP5 protein was functionally active because AQP5 expression resulted in a significant increase in the osmotically directed net fluid rate across monolayers of NS-SV-DC cells. By the analysis of bisulfite sequencing of CpG islands in the AQP5 promoter, hypermethylation within the consensus Sp1-binding sites was commonly observed in parental cell clones, whereas demethylation at the CGs, one in the second consensus Sp1 element and the other outside of the third consensus Sp1 element in the AQP5 promoter, was detected in NS-SV-DC cells after treatment with 5-Aza-CdR. By analyzing the luciferase activity of transfected AQP5 promoter vectors, it became evident that demethylation at the CGs cooperatively functions between these two sites to induce AQP5 expression. Our data, therefore, suggest that treatment of ductal cells with 5-Aza-CdR could result in the expression of the AQP5 gene, thereby leading to increased fluid secretion from ductal cells in SS salivary glands.     

Regulatory B cells from hilar lymph nodes of tolerant mice in a murine model of allergic airway disease are CD5+, express TGF-β, and co-localize with CD4+Foxp3+ T cells

In a biphasic, ovalbumin (OVA)-induced murine asthma model where allergic airway disease is followed by resolution and the development of local inhalational tolerance (LIT), transforming growth factor (TGF)-β-expressing CD5⁺ B cells were selectively expanded locally in hilar lymph nodes (HLN) of LIT mice. LIT HLN CD5⁺ B cells, but not LIT HLN CD5^– B cells, induced expression of Foxp3 in CD4⁺CD25^– T cells in vitro. These CD5⁺ regulatory B cells (Breg) and CD4⁺Foxp3⁺ T cells demonstrated similar increases in expression of chemokine receptors (CXCR4 and CXCR5) and co-localized in HLN B cell zones of LIT mice. The adoptive transfer of LIT HLN CD5⁺ B cells, but not LIT HLN CD5^– B cells, increased the number of CD4⁺Foxp3⁺ T cells in the lung and inhibited airway eosinophilia in this OVA model. Thus, Breg in HLNs of LIT mice reside in a CD5⁺ TGF-β-producing subpopulation and co-localize with CD4⁺Foxp3⁺ T cells.