Isolation and culture of mouse intestinal cells.

Complex cell signal transduction mechanisms regulate intestinal epithelial shape, polarity, motility, organelles, cell membrane components as well as physical and mechanical properties to influence alimentary digestion, absorption, secretion, detoxification and fluid balance. Interactions between the epithelial cells and adjacent mesenchyme are central to intestinal homeostasis although the key regulatory molecules of specific differentiation steps remain unclear. Isolation and primary culture of heterotypic murine intestinal cells provides a model system for elucidation of essential molecular cross-talk between epithelium and mesenchyme that may provide several biological and practical advantages over transformed cell lines. An in vitro primary culture system for neonatal rat or mouse intestinal cells has been established that forms monolayers, expresses intestine-specific epithelial features including intestinal brush borders and appropriate hydrolase enzymes. Our studies confirm the promise of this method which may advance our understanding of heterotypic cellular interactions implicated in intestinal function and may provide important insights into the pathobiology of disease.

Early target cells of measles virus after aerosol infection of non-human primates

Measles virus (MV) is highly infectious, and has long been thought to enter the host by infecting epithelial cells of the respiratory tract. However, epithelial cells do not express signaling lymphocyte activation molecule (CD150), which is the high-affinity cellular receptor for wild-type MV strains. We have generated a new recombinant MV strain expressing enhanced green fluorescent protein (EGFP), based on a wild-type genotype B3 virus isolate from Khartoum, Sudan (KS). Cynomolgus macaques were infected with a high dose of rMV(KS)EGFP by aerosol inhalation to ensure that the virus could reach the full range of potential target cells throughout the entire respiratory tract. Animals were euthanized 2, 3, 4 or 5 days post-infection (d.p.i., n?=?3 per time point) and infected (EGFP(+)) cells were identified at all four time points, albeit at low levels 2 and 3 d.p.i. At these earliest time points, MV-infected cells were exclusively detected in the lungs by fluorescence microscopy, histopathology and/or virus isolation from broncho-alveolar lavage cells. On 2 d.p.i., EGFP(+) cells were phenotypically typed as large mononuclear cells present in the alveolar lumen or lining the alveolar epithelium. One to two days later, larger clusters of MV-infected cells were detected in bronchus-associated lymphoid tissue (BALT) and in the tracheo-bronchial lymph nodes. From 4 d.p.i. onward, MV-infected cells were detected in peripheral blood and various lymphoid tissues. In spite of the possibility for the aerosolized virus to infect cells and lymphoid tissues of the upper respiratory tract, MV-infected cells were not detected in either the tonsils or the adenoids until after onset of viremia. These data strongly suggest that in our model MV entered the host at the alveolar level by infecting macrophages or dendritic cells, which traffic the virus to BALT or regional lymph nodes, resulting in local amplification and subsequent systemic dissemination by viremia.

Inactivation of Rb in stromal fibroblasts promotes epithelial cell invasion

Stromal-derived growth factors are required for normal epithelial growth but are also implicated in tumour progression. We have observed inactivation of the retinoblastoma protein (Rb), through phosphorylation, in cancer-associated fibroblasts in oro-pharyngeal cancer specimens. Rb is well known for its cell-autonomous effects on cancer initiation and progression; however, cell non-autonomous functions of Rb are not well described. We have identified a cell non-autonomous role of Rb, using three-dimensional cultures, where depletion of Rb in stromal fibroblasts enhances invasive potential of transformed epithelia. In part, this is mediated by upregulation of keratinocyte growth factor (KGF), which is produced by the depleted fibroblasts. KGF drives invasion of epithelial cells through induction of MMP1 expression in an AKT- and Ets2-dependent manner. Our data identify that stromal fibroblasts can alter the invasive behaviour of the epithelium, and we show that altered expression of KGF can mediate these functions. © European Molecular Biology Organization.

Airway epithelial cell apoptosis and inflammation in COPD, smokers and non-smokers

We hypothesised that primary bronchial epithelial cells (PBECs) from subjects with COPD respond differently to Pseudomonas aeruginosa lipopolysaccharide (PA LPS) after cigarette smoke extract (CSE) exposure than PBECs obtained from smokers without airflow obstruction (SWAO) and non-smokers (NS).PBECs from 16 COPD subjects, 10 SWAOand 9 NS were cultured at air-liquid interface. Cultures were incubated with CSE prior to stimulation with PA LPS. IL-6 and IL-8 were measured by ELISA and Toll-like receptor 4 expression by FACS. Activation of NF-?B was determined by western blotting and ELISA, and MAPK and caspase-3 activity by western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) methods.Constitutive release of IL-8 and IL-6 was greatest from the COPD cultures.However, CSE pre-treatment followed by PA LPS stimulation reduced IL-8 release from COPD PBECs, but increased it from cells of SWAOand NS. TLR-4 expression,MAPK and NF-?B activation in COPD cultures were reduced after CSE treatment, but not in the SWAOor NS groups, which was associated with increased apoptosis.CSE attenuates inflammatory responses to LPS in cells from people with COPD but not those from non-smoking individuals and those who smoke without airflow obstruction.

The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

Shigella flexneri causes bacillary dysentery in humans. Essential to the establishment of the disease is the invasion of the colonic epithelial cells. Here we investigated the role of the lipopolysaccharide (LPS) O antigen in the ability of S. flexaeri to adhere to and invade polarized Caco-2 cells. The S. flexneri 2a O antigen has two preferred chain lengths: a short O antigen (S-OAg) regulated by the WzzB protein and a very long O antigen (VL-OAg) regulated by Wzz(pHS2). Mutants with defined deletions of the genes required for O-antigen assembly and polymerization were constructed and assayed for their abilities to adhere to and enter cultured epithelial cells. The results show that both VL- and S-OAg are required for invasion through the basolateral cell membrane. In contrast, the absence of O antigen does not impair adhesion. Purified LPS does not act as a competitor for the invasion of Caco-2 cells by the wild-type strain, suggesting that LPS is not directly involved in the internalization process by epithelial cells.

Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology

Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.

Participation of adult mouse bone marrow cells in reconstitution of skin

Results of recent studies have indicated that bone marrow cells can differentiate into various cells of ectodermal, mesodermal, and endodermal origins when transplanted into the body. However, the problems associated with those experiments such as the long latent period, rareness of the event, and difficulty in controlling the processes have hampered detailed mechanistic studies. In the present study, we examined the potency of mouse bone marrow cells to differentiate into cells comprising skin tissues using a skin reconstitution assay. Bone marrow cells from adult green fluorescent protein (GFP)-transgenic mice were transplanted in a mixture of embryonic mouse skin cells (17.5 days post-coitus) onto skin defects made on the backs of nude mice. Within 3 weeks, fully differentiated skin with hair was reconstituted. GFP-positive cells were found in the epidermis, hair follicles, sebaceous glands, and dermis. The localization and morphology of the cells, results of immunohistochemistry, and results of specific staining confirmed that the bone marrow cells had differentiated into epidermal keratinocytes, sebaceous gland cells, follicular epithelial cells, dendritic cells, and endothelial cells under the present conditions. These results indicate that this system is suitable for molecular and cellular mechanistic studies on differentiation of stem cells to various epidermal and dermal cells.

Limbal stem cells of the corneal epithelium

Stem cells have certain unique characteristics, which include longevity, high capacity of self-renewal with a long cell cycle time and a short S-phase duration, increased potential for error-free proliferation, and poor differentiation. The ocular surface is made up of two distinct types of epithelial cells, constituting the conjunctival and the corneal epithelia. Although anatomically continuous with each other at the corneoscleral limbus, the two cell phenotypes represent quite distinct subpopulations. Stem cells for the cornea reside at the corneoscleral limbus. The limbal palisades of Vogt and the interpalisade rete ridges are believed to be repositories of stem cells. The microenvironment of the limbus is considered to be important in maintaining the stemness of stem cells. Limbal stem cells also act as a 'barrier' to conjunctival epithelial cells and normally prevent them from migrating on to the corneal surface. Under certain conditions, however, the limbal stem cells may be partially or totally depleted, resulting in varying degrees of stem cell deficiency with resulting abnormalities in the corneal surface. Such deficiency of limbal stem cells leads to 'conjunctivalization' of the cornea with vascularization, appearance of goblet cells, and an irregular and unstable epithelium. This results in ocular discomfort and reduced vision. Partial stem cell deficiency can be managed by removing the abnormal epithelium and allowing the denuded cornea, especially the visual axis, to resurface with cells derived from the remaining intact limbal epithelium. In total stem cell deficiency, autologous limbus from the opposite normal eye or homologous limbus from living related or cadaveric donors can be transplanted on to the affected eye. With the latter option, systemic immunosuppression is required. Amniotic membrane transplantation is a useful adjunct to the above procedures in some instances. Copyright (C) 2000 Elsevier Science Inc.

Lipoxins attenuate renal fibrosis by inducing let-7c and suppressing TGFβR1

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-ß1-induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-ß1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-ß1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-ß1 signaling pathway, including the TGF-ß receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-ß receptor type 1 and the response to TGF-ß1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-ß1-induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in severe asthma

BACKGROUND: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated.

OBJECTIVE: In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways.

METHODS: Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1.

RESULTS: Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current.

CONCLUSIONS: Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.

Keratinocyte Growth-Factor Promotes Epithelial Survival and Resolution in a Human Model of Lung Injury

Rationale: Increasing epithelial repair and regeneration may hasten resolution of lung injury in patients with the Acute Respiratory Distress Syndrome (ARDS). In animal models of ARDS, Keratinocyte Growth Factor (KGF) reduces injury and increases epithelial proliferation and repair. The effect of KGF in the human alveolus is unknown.

Objectives: To test whether KGF can attenuate alveolar injury in a human model of ARDS.

Methods: Volunteers were randomized to intravenous KGF (60 μg/kg) or placebo for 3 days, before inhaling 50μg lipopolysaccharide. Six hours later, subjects underwent bronchoalveolar lavage (BAL) to quantify markers of alveolar inflammation and cell-specific injury.

Measurements and Main Results: KGF did not alter leukocyte infiltration or markers of permeability in response to LPS. KGF increased BAL concentrations of Surfactant Protein D (SP-D), MMP-9, IL-1Ra, GM-CSF and CRP. In vitro, BAL fluid from KGF-treated subjects (KGF BAL) inhibited pulmonary fibroblast proliferation, but increased alveolar epithelial proliferation. Active MMP-9 increased alveolar epithelial wound repair. Finally, BAL from the KGF pre-treated group enhanced macrophage phagocytic uptake of apoptotic epithelial cells and bacteria compared with BAL from the placebo-treated group. This effect was blocked by inhibiting activation of the GM-CSF receptor.

Conclusions: KGF treatment increases BAL SP-D, a marker of type II alveolar epithelial cell proliferation in a human model of ALI. Additionally KGF increases alveolar concentrations of the anti-inflammatory cytokine IL-1Ra, and mediators that drive epithelial repair (MMP-9) and enhance macrophage clearance of dead cells and bacteria (GM-CSF).

SMAD inhibition attenuates epithelial to mesenchymal transition by primary keratinocytes in vitro

Epithelial to mesenchymal transition (EMT) is a process whereby epithelial cells undergo transition to a mesenchymal phenotype and contribute directly to fibrotic disease. Recent studies support a role for EMT in cutaneous fibrotic diseases including scleroderma and hypertrophic scarring, though there is limited data on the cytokines and signalling mechanisms regulating cutaneous EMT. We investigated the ability of TGF-β and TNF-α, both over-expressed in cutaneous scleroderma and central mediators of EMT in other epithelial cell types, to induce EMT in primary keratinocytes and studied the signalling mechanisms regulating this process. TGF-β induced EMT in normal human epidermal keratinocytes (NHEK cells) and this process was enhanced by TNF-α. EMT was characterised by changes in morphology, proteome (down-regulation of E-cadherin and Zo-1, and up-regulation of vimentin and fibronectin), MMP secretion and COL1α1 mRNA expression. TGF-β and TNF-α in combination activated SMAD and p38 signalling in NHEK cells. P38 inhibition with SB203580 partially attenuated EMT, whereas SMAD inhibition using SB431542 significantly inhibited EMT and also reversed established EMT. These data highlight the retained plasticity of adult keratinocytes and support further studies of EMT in clinically relevant in vivo models of cutaneous fibrosis, and investigation of SMAD inhibition as a potential therapeutic intervention. This article is protected by copyright. All rights reserved.

Gremlin1 preferentially binds to Bone Morphogenetic Protein-2 (BMP-2) and BMP-4 over BMP-7

Gremlin (Grem1) is a member of the DAN family of secreted bone morphogenetic protein (BMP) antagonists. Bone morphogenetic protein-7 (BMP-7) mediates protective effects during renal fibrosis-associated with diabetes and other renal diseases. The pathogenic mechanism of Grem1 during DN has been suggested to be binding and inhibition of BMP-7. However, the precise interactions between Grem1, BMP-7 and other BMPs have not been accurately defined. Here we show the affinity of Grem1 for BMP-7 is lower than that of BMP-2 and BMP-4, using a combination of surface plasmon resonance and cell culture techniques. Using kidney proximal tubule cells and HEK-293 cell Smad1/5/8 phosphorylation and BMP-dependent gene expression as readout, Grem1 consistently demonstrated a higher affinity for BMP-2>4>7. Cell-associated Grem1 did not inhibit BMP-2 or BMP-4 mediated signalling, suggesting that Grem1-BMP-2 binding occurred in solution, preventing BMP receptor activation. These data suggest that Grem1 preferentially binds to BMP-2 and this may be the dominant complex in a disease situation where levels of Grem1 and BMPs are elevated.

IHG-1 increases mitochondrial fusion and bioenergetic function

Induced in high glucose-1 (IHG-1) is a conserved mitochondrial protein associated with diabetic nephropathy (DN) that amplifies profibrotic transforming growth factor (TGF)-β1 signaling and increases mitochondrial biogenesis. Here we report that inhibition of endogenous IHG-1 expression results in reduced mitochondrial respiratory capacity, ATP production, and mitochondrial fusion. Conversely, overexpression of IHG-1 leads to increased mitochondrial fusion and also protects cells from reactive oxygen species-induced apoptosis. IHG-1 forms complexes with known mediators of mitochondrial fusion-mitofusins (Mfns) 1 and 2-and enhances the GTP-binding capacity of Mfn2, suggesting that IHG-1 acts as a guanine nucleotide exchange factor. IHG-1 must be localized to mitochondria to interact with Mfn1 and Mfn2, and this interaction is necessary for increased IHG-1-mediated mitochondrial fusion. Together, these findings indicate that IHG-1 is a novel regulator of both mitochondrial dynamics and bioenergetic function and contributes to cell survival following oxidant stress. We propose that in diabetic kidney disease increased IHG-1 expression protects cell viability and enhances the actions of TGF-β, leading to renal proximal tubule dedifferentiation, an important event in the pathogenesis of this devastating condition.