Age-related expression of MCP-1 and MMP-3 in mouse intervertebral disc in relation to TWEAK and TNF-α stimulation.

This study was undertaken to investigate the age-related differences of monocyte chemotactic protein-1 (MCP-1) and matrix metalloproteinase-3 (MMP-3) expression in mouse intervertebral disc (IVD) and to determine whether MMP-3 plays a role in disc degeneration. Expression of MCP-1 and MMP-3 mRNA in mouse IVD was assessed by quantitative PCR. The ability of MCP-1 and MMP-3 expression in IVD to respond to TNF-α or TWEAK stimulation was examined by quantitative PCR, WB, ELISA, and immunohistochemistry. IVD derived from MMP-3-deficient and wild-type mice were compared using Safranin-O staining and immunohistochemistry. mRNA levels of MCP-1 and MMP-3 in IVD significantly diminished and the ability of MCP-1 or MMP-3 expression to respond to TNF-α or TWEAK stimulation was significantly reduced as age increased. IVD derived from 64-week-old wild-type mice showed clearly diffuse proteoglycan loss by Safranin-O staining and immunohistochemistry compared with younger mice. However, no loss of proteoglycan and typeII collagen were observed in IVD derived from 64-week-old MMP-3-deficient mice. MCP-1 and MMP-3 expression in mouse IVD showed age-related decreases. The response to inflammation in IVD also displayed age-related changes. Therefore, disc degeneration may vary with the patients' age and targeting MMP-3 may be a possible future therapeutic strategy for disc degeneration.

TGF-beta signaling may play a role in the development of goblet cell hyperplasia in a mouse model of allergic rhinitis.

BACKGROUND: Transforming growth factor-beta (TGF-beta) levels are elevated in the nasal mucosa in allergic rhinitis. However, because TGF-beta is secreted extracellulary in latent complexes, it remains unclear whether the local TGF-beta expression actually drives active signaling and affects the pathophysiology of allergic rhinitis. The objective of this study is to investigate whether TGF-beta signaling is activated in allergic rhinitis and plays a role in the pathophysiology of allergic rhinitis. METHODS: An ovabumin (OVA)-sensitized and -nasally challenged mouse model of allergic rhinitis was established and phosphorylation of Smad2 in the nasal mucosa was examined by immunohistochemistry. In addition, the effects of the pharmacological inhibition of endogenous TGF-beta signaling on the allergic rhinitis model were histologically examined. Furthermore, phosphorylation of Smad2 in the nasal mucosa samples obtained from patients with allergic rhinitis was also evaluated. RESULTS: In the mouse model of allergic rhinitis, OVA challenge induced phosphorylation of Smad2 predominantly in epithelial cells in the nasal mucosa. In addition, the administration of an inhibitor of TGF-beta type I receptor kinase activity during OVA challenge suppressed goblet cell hyperplasia in the nasal mucosa. Furthermore, phosphorylated Smad2 expression increased in nasal epithelial cells in patients with allergic rhinitis. CONCLUSIONS: These results suggest that TGF-beta signaling is activated in epithelial cells in the nasal mucosa in allergic rhinitis and may contribute to the development of goblet cell hyperplasia.

Thymic stromal lymphopoietin is a critical mediator of IL-13-driven allergic inflammation.

Both thymic stromal lymphopoietin (TSLP) and IL-13 are essential cytokines for the development of allergic inflammation. However, a causal link between TSLP and IL-13 has not yet been fully elucidated. This study aimed to investigate whether IL-13 induces TSLP expression and whether the induction contributes to the development of allergic inflammation. We found that IL-13 induced TSLP expression in mouse nasal tissue specimens in a Stat6-dependent manner. In addition, intranasal challenge of mice with IL-13 induced TSLP expression in the nasal epithelium. Importantly, intranasal IL-13 challenge induced eosinophilia and goblet cell hyperplasia in the nasal mucosa in mice, which was inhibited by the blockade of TSLP activity with anti-TSLP Ab. These findings suggest that TSLP is an important mediator of IL-13-driven allergic inflammation in the nasal mucosa. Taken together with the recent findings that IL-13 is a critical downstream element for TSLP-driven allergic inflammation, TSLP may function both upstream and downstream of IL-13, thus providing an additional rationale as to why TSLP plays such a central role in the development of allergic inflammation.

A subcytotoxic dose of subtilase cytotoxin prevents lipopolysaccharide-induced inflammatory responses, depending on its capacity to induce the unfolded protein response.

Subtilase cytotoxin (SubAB) is the prototype of a newly identified family of AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. SubAB specifically cleaves the essential endoplasmic reticulum (ER) chaperone BiP (GRP78), resulting in the activation of ER stress-induced unfolded protein response (UPR). We have recently shown that the UPR following ER stress can suppress cellular responses to inflammatory stimuli during the later phase, in association with inhibition of NF-kappaB activation. These findings prompted us to hypothesize that SubAB, as a selective UPR inducer, might have beneficial effects on inflammation-associated pathology via a UPR-dependent inhibition of NF-kappaB activation. The pretreatment of a mouse macrophage cell line, RAW264.7, with a subcytotoxic dose of SubAB-triggered UPR and inhibited LPS-induced MCP-1 and TNF-alpha production associated with inhibition of NF-kappaB activation. SubA(A272)B, a SubAB active site mutant that cannot induce UPR, did not show such effects. In addition, pretreatment with a sublethal dose of SubAB, but not SubA(A272)B, protected the mice from LPS-induced endotoxic lethality associated with reduced serum MCP-1 and TNF-alpha levels and also prevented the development of experimental arthritis induced by LPS in mice. Collectively, although SubAB has been identified originally as a toxin associated with the pathogenesis of hemolytic uremic syndrome, the unique ability of SubAB to selectively induce the UPR may have the potential to prevent LPS-associated inflammatory pathology under subcytotoxic conditions.

The latent form of transforming growth factor-beta administered orally is activated by gastric acid in mice.

Transforming growth factor-beta (TGFbeta) is abundant in mammalian milk in a latent form. However, whether the latent form of TGFbeta in human milk is converted to the active form in vivo remains uncertain. To address this issue, we first investigated whether latent TGFbeta or human milk-borne latent TGFbeta was activated in an in vitro assay, simulating the effects of gastric acid. We then tested whether gastric acid was necessary for the activation of orally administered latent TGFbeta or human milk-borne latent TGFbeta in mice by inhibiting gastric acidity with cimetidine, an antagonist of H2-receptors. Latent TGFbeta or human milk-borne latent TGFbeta increased Smad-responsive promoter activity in MFB-F11 reporter cells at pH 1.2, but not at pH 7.0, regardless of the presence or absence of the gastric protease pepsin. In mice treated orally with latent TGFbeta (5 microg/mouse), the phosphorylation of Smad2 and TGFbeta target gene mRNA expression (TGFbeta and Smad7) was increased in the small intestine (P < 0.05) and this effect was inhibited by cimetidine (100 mg/kg, intraperitoneally). Similarly, mice treated orally with 1200 microL/d of human milk containing latent TGFbeta (3 microg/L) for 2 wk had increased TGFbeta and Smad7 mRNA expression in the small intestine (P < 0.05) and this was inhibited by the antiacid treatment. Therefore, the latent form of TGFbeta, such as TGFbeta in human milk, can be activated by gastric acid following oral administration in mice. This process may be involved in the conversion of human milk-borne latent TGFbeta to the active form in vivo.

House dust mite allergen Der f 1 can induce the activation of latent TGF-beta via its protease activity.

A major house dust mite allergen Der f 1 belongs to the papain-like cysteine protease family. This study investigated whether Der f 1 can cleave the latency-associated peptide (LAP) of transforming growth factor (TGF)-beta via its proteolytic activity and activate latent TGF-beta. We found that Der f 1 can cleave LAP and induce the activation of latent TGF-beta, leading to functional Smad signaling. Importantly, these actions of Der f 1 were inhibited by cysteine protease inhibitor E64 or inactivation of the protease activity by heat. Thus, latent TGF-beta may be a direct target of Der f 1 protease activity.

Transforming growth factor-beta activity in commercially available pasteurized cow milk provides protection against inflammation in mice.

Cow milk contains a large amount of an immunoregulatory cytokine, transforming growth factor-beta (TGFbeta). The present study investigated whether commercially available pasteurized cow milk retains TGFbeta activity both in vitro and in vivo. Some commercial cow milk increased TGFbeta/Smad-responsive reporter activity and induced Smad2 phosphorylation and the transcription of the TGFbeta/Smad target genes TGFbeta itself and Smad7 in vitro. Mice treated orally with 500 microL of cow milk containing TGFbeta (3 microg/L) daily for 2 wk had increased phosphorylation of Smad2 and TGFbeta and Smad7 mRNA expression in the intestine. These mice also had significantly greater serum TGFbeta concentrations than the mice treated orally with PBS. Furthermore, oral administration of 500 microL of cow milk containing TGFbeta (3 microg/L) daily for 2 wk before the induction of dextran sodium sulfate colitis and lipopolysaccharide-induced endotoxemia ameliorated tissue damage and mortality, respectively, in mice. These in vivo effects of cow milk were abrogated by the simultaneous administration of TGFbeta type I receptor kinase inhibitor with the cow milk, and they were not observed after the oral administration of cow's milk containing little TGFbeta. In humans, 1 oral challenge of 10 mL/kg cow milk containing TGFbeta (3 microg/L) increased the plasma TGFbeta concentrations at 4 h after the challenge. Thus, some commercially available pasteurized cow milk retains TGFbeta activity, which may be able to provide protection against experimental colitis and endotoxemia associated with increased intestinal and circulating TGFbeta levels.

A potential role of thymic stromal lymphopoietin in the recruitment of macrophages to mouse intervertebral disc cells via monocyte chemotactic protein 1 induction: implications for herniated discs.

OBJECTIVE: To determine whether thymic stromal lymphopoietin (TSLP) plays a role in the resorption of herniated disc tissue. METHODS: The expression of TSLP messenger RNA (mRNA) and protein in mouse intervertebral disc cells was assessed by quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical analysis. The ability of mouse intervertebral disc cells to respond to TSLP stimulation was examined by Western blot analysis, ELISA, and protein array analysis. Intracellular signaling pathways involved in TSLP signaling in mouse intervertebral disc cells were investigated using several chemical inhibitors. The role of TSLP in macrophage migration into the intervertebral disc was assessed by in vitro migration assay. Finally, TSLP expression in clinical specimens derived from patients with a herniated disc was examined by immunohistochemistry. RESULTS: Mouse intervertebral disc cells expressed TSLP mRNA and protein upon stimulation with NF-kappaB-activating ligands such as tumor necrosis factor alpha. In addition, the mouse intervertebral disc cells expressed the TSLP receptor and produced monocyte chemotactic protein 1 (MCP-1; CCL2) and macrophage colony-stimulating factor in response to TSLP stimulation. Both anulus fibrosus and nucleus pulposus intervertebral disc cells expressed MCP-1 upon TSLP stimulation, which was mediated via the phosphatidylinositol 3-kinase/Akt pathway. Consistently, the supernatants of TSLP-activated intervertebral disc cultures had the capacity to induce macrophage migration in an MCP-1-dependent manner. Finally, TSLP and MCP-1 were coexpressed in human herniated disc specimens in which macrophage infiltration into the tissue was observed. CONCLUSION: TSLP induced by NF-kappaB-activating ligands in intervertebral discs may contribute to the recruitment of macrophages to the intervertebral disc by stimulating MCP-1 production and may be involved in the resorption of herniated disc tissue.

Cigarette smoke extract induces thymic stromal lymphopoietin expression, leading to T(H)2-type immune responses and airway inflammation.

BACKGROUND: Both active and passive smoking are considered to be risk factors for asthma development. However, the precise mechanisms involved remain elusive. Recently, thymic stromal lymphopoietin (TSLP) has been shown to play a key role in the development of T(H)2-type allergic inflammation in patients with asthma. OBJECTIVE: The aim of this study was to investigate whether there was a causal relationship between cigarette smoke exposure and TSLP expression in the lung. METHODS: We examined the effects of repeated intranasal exposure of cigarette smoke extract (CSE) on TSLP mRNA and protein expression in the mouse lung by means of real-time PCR, Western blotting, and immunohistochemistry. We also examined the effects of intranasal exposure of CSE plus ovalbumin (OVA) on T(H)2-type immune responses and lung pathology. RESULTS: Repeated exposure of CSE induced TSLP mRNA and protein expression, which was inhibited by treatment with antioxidative N-acetylcysteine and by TNF-alpha receptor I deficiency. In addition, the intranasal exposure of CSE simultaneously with OVA induced OVA-specific T(H)2-type immune responses and airway inflammation, which were inhibited by the blockade of the TSLP activity. CONCLUSION: CSE induced TSLP expression in the mouse lung in an oxidative stress-dependent and TNF-alpha receptor I-dependent manner, and when challenged simultaneously with an antigen, CSE promoted the development of airway inflammation in association with T(H)2-type immune responses.

Mast cell regulation of epithelial TSLP expression plays an important role in the development of allergic rhinitis.

Epithelial cell-derived thymic stromal lymphopoietin (TSLP) is a master switch for asthma or atopic dermatitis by inducing a dendritic cell-mediated Th2-type allergic inflammation. Allergic rhinitis is also pathologically characterized by Th2-type allergic inflammation. This study demonstrates that mast cells regulate the epithelial TSLP expression in allergic rhinitis. TSLP expression was found to be up-regulated predominantly in the nasal epithelium in the ovalbumin (OVA)-sensitized and -nasally challenged mouse model of allergic rhinitis, which was abolished in mast cell-deficient WBB6F1-W/W(v) in comparison with control WBB6F1-+/+ mice. Similarly, the epithelial TSLP expression was reduced in Fc receptor gamma chain (FcgammaR)-deficient mice, where the high-affinity IgE receptor (FcepsilonRI) is not expressed on mast cells, in comparison with control C57BL/6 mice. Furthermore, the administration of neutralizing TSLP antibody during the challenge phase of OVA inhibited the development of allergic rhinitis. These results suggest that the direct stimulation of epithelial cells by antigens alone may not be sufficient to induce TSLP expression in the nasal epithelium, and that mast cell regulation of epithelial TSLP expression, possibly via FcepsilonRI, plays an important role in the development of allergic rhinitis.

T cell receptor-mediated signaling induces GRP78 expression in T cells: the implications in maintaining T cell viability.

The 78-kDa glucose-regulated protein (GRP78) is an important molecular chaperone in the endoplasmic reticulum (ER) induced by various stresses. This study showed that stimulation with anti-CD3 mAb, PMA plus ionomycin, or an antigen increased the levels of GRP78 mRNA in primary T cells, which was inhibited by Ca(2+) chelators EGTA and BAPTA-AM and by an inhibitor of calcineurin FK506. In addition, the specific knockdown of GRP78 protein expression induced apoptosis in mouse EL-4 T cell line associated with CHOP induction and caspase-3 activation. Furthermore, overexpression of GRP78 inhibited PMA/ionomycin-induced cell death in EL-4 cells. Collectively, GRP78 expression is induced by TCR activation via a Ca(2+)-dependent pathway and may play a critical role in maintaining T cell viability in the steady and TCR-activated states. These results suggest a novel regulatory mechanism and an essential function of GRP78 in T cells.

A possible link between resveratrol and TGF-beta: resveratrol induction of TGF-beta expression and signaling.

Resveratrol, a polyphenolic compound found in the skin of red fruits, exhibits anti-inflammatory, anti-oxidative, and anti-proliferative characteristics. Transforming growth factor-beta (TGF-beta) is a pleiotropic cytokine that also displays such properties. We therefore hypothesized that there might be a functional link between resveratrol and TGF-beta. This study reports that resveratrol increased transcription of the TGF-beta2 gene, enhanced the production of TGF-beta2 protein, and activated Smad signaling in an autocrine manner in A549 human lung epithelial cell line. Thus, some of the beneficial effects of resveratrol on human health might be mediated, in part, through its effects on TGF-beta expression and signaling.

Thymic stromal lymphopoietin secretion of synovial fibroblasts is positively and negatively regulated by Toll-like receptors/nuclear factor-kappaB pathway and interferon-gamma/dexamethasone.

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine produced by epithelial cells and triggers dendritic cell-mediated Th2 type allergic inflammatory responses. This study investigated whether Toll-like receptor (TLR) ligands, lipopolysaccharide (LPS) and poly-IC affect TSLP production in synovial fibroblasts. Enzyme-linked immunosorbent assay showed that LPS and poly-IC upregulated TSLP production in synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). In addition, we found that nuclear factor (NF)-kappaB inhibitor IMD-0354, dexamethasone, and interferon (IFN)-gamma inhibited the LPS- and poly-IC-induced TSLP production in RA and OA synovial fibroblasts. Thus, LPS and poly-IC can upregulate TSLP via a NF-kappaB pathway in synovial fibroblasts, which is downregulated by dexamethasone and interferon (IFN)-gamma. The current findings suggest that TSLP may be involved in the pathophysiology of inflammatory arthritis as well as allergic disease.

Orally administered TGF-beta is biologically active in the intestinal mucosa and enhances oral tolerance.

BACKGROUND: Epidemiologic studies suggest that TGF-beta in breast milk provides protection against allergic disease during infancy. However, it is unclear whether orally administered TGF-beta, such as TGF-beta in human milk, retains and exerts its activity in the intestinal mucosa and can affect immune response (tolerance) to dietary antigens. OBJECTIVE: We sought to determine whether orally administered TGF-beta is biologically active in intestinal mucosa and affects oral tolerance. METHODS: Activity of orally administered TGF-beta in the intestinal mucosa was evaluated by means of in vivo imaging with transgenic mice expressing a Smad-responsive reporter construct (SBE-luc mice), by means of immunohistochemical staining with anti-phosphorylated Smad2 antibody, and by means of real-time RT-PCR analysis of TGF-beta and Smad7 mRNA expression. The effects of orally administered TGF-beta on oral tolerance induction were assessed in mice tolerized by means of high-dose ovalbumin (OVA) feeding. RESULTS: The oral administration of TGF-beta increased Smad-responsive reporter activity in the intestines of SBE-luc mice and induced Smad2 phosphorylation and TGF-beta and Smad7 mRNA expression in the intestines of BALB/c mice. Serum TGF-beta levels were also increased after oral administration of TGF-beta. BALB/c mice treated orally with OVA and TGF-beta showed augmented reduction of OVA-specific IgE and IgG1 antibodies, T-cell reactivity, and immediate-type skin reactions when compared with the mice treated orally with OVA alone. CONCLUSIONS: Orally administered TGF-beta retains sufficient biologic activity in intestinal mucosa and enhances oral tolerance. CLINICAL IMPLICATIONS: Oral administration of TGF-beta might become a potential strategy to prevent allergic diseases, such as food allergy.

A possible role for TSLP in inflammatory arthritis.

Thymic stromal lymphopoietin (TSLP) is an IL-7-like cytokine that triggers dendritic cell-mediated Th2-type inflammatory responses and is considered as a master switch for allergic inflammation. In this study, we found increased levels of TSLP and, also TNF-alpha as previously reported, in synovial fluid specimens derived from patients with rheumatoid arthritis (RA) when compared with those from patients with osteoarthritis (OA). In addition, TNF-alpha up-regulated TSLP expression in RA- and OA-derived synovial fibroblasts, which was inhibited by IFN-gamma. Furthermore, anti-TSLP neutralizing antibody ameliorated a TNF-alpha-dependent experimental arthritis induced by anti-type II collagen antibody in mice. Collectively, these results suggest that TSLP, as a downstream molecule of TNF-alpha, may be involved in the pathophysiology of inflammatory arthritis. TSLP might thus play a role not only in allergic diseases but also in inflammatory arthritis such as RA.