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David R Garrod

Publications and source records attributed to David R Garrod.

12 recordsLinked to original sources
PubMed2006

Calcium-independent desmosomes of keratinocytes are hyper-adhesive.

Desmosomes in tissues are resistant to disruption by chelation of extracellular calcium. It has been suggested that this represents a hyper-adhesive state of these intercellular junctions that is crucial for the maintenance of epidermal integrity. Desmosomes change to a lower affinity, calcium-dependent adhesive state when cells are cultured at low density or when an intact epithelial cell sheet is wounded. Here we demonstrate that cells of the immortalized human keratinocyte line HaCaT acquire calcium-independent desmosomes in confluent culture. An adhesion assay shows that HaCaT cells with calcium-independent desmosomes are more cohesive than cells with calcium-dependent desmosomes. This assay relates directly to desmosomal adhesion because it involves splitting of the desmosomal adhesive material. Moreover, the difference in adhesiveness between calcium-dependent and calcium-independent desmosomes involves no quantitative change in the known protein composition of desmosomes. Instead, switching between the two adhesive states can be achieved by activation or inhibition of protein kinase C (PKC), suggesting a direct effect of PKC signalling on desmosomal adhesion. These results provide direct support for the concept of hyper-adhesiveness in desmosomes.

Calcium↗
PubMed2005

Hyper-adhesion in desmosomes: its regulation in wound healing and possible relationship to cadherin crystal structure.

The resistance of tissues to physical stress is dependent upon strong cell-cell adhesion in which desmosomes play a crucial role. We propose that desmosomes fulfil this function by adopting a more strongly adhesive state, hyper-adhesion, than other junctions. We show that the hyper-adhesive desmosomes in epidermis resist disruption by ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) and are thus independent of Ca2+. We propose that Ca2+ independence is the normal condition for tissue desmosomes. Ca2+ independence is associated with an organised arrangement of the intercellular adhesive material exemplified by a dense midline. When epidermis is wounded, desmosomes in the wound-edge epithelium lose hyper-adhesiveness and become Ca2+ dependent, i.e. readily dissociated by EGTA. Ca2+-dependent desmosomes lack a midline and show narrowing of the intercellular space. We suggest that this indicates a less-organised, weakly adhesive arrangement of the desmosomal cadherins, resembling classical cadherins in adherens junctions. Transition to Ca2+ dependence on wounding is accompanied by relocalisation of protein kinase C alpha to desmosomal plaques suggesting that an 'inside-out' transmembrane signal is responsible for changing desmosomal adhesiveness. We model hyper-adhesive desmosomes using the crystal packing observed for the ectodomain of C-cadherin and show how the regularity of this 3D array provides a possible explanation for Ca2+ independence.

Animals↗
PubMed2005

Interferon-gamma selectively increases epithelial permeability to large molecules by activating different populations of paracellular pores.

Impairment of the gut epithelial barrier by agents such as IFNgamma may play a key role in the pathogenesis of inflammatory disorders by increasing the paracellular penetration of luminal macromolecules, potentially including bacterial antigens. Owing to limitations of current paracellular probes, little is known about the precise functional changes induced by IFNgamma and how these relate to the development of increased macromolecular permeability. Here we investigate how IFNgamma modulates this pathway in T84 monolayers using a novel profiling technique that resolves different populations of paracellular pores by simultaneous analysis of 24 permeability probes of defined molecular size. Two types of functional pore present in control monolayers, an abundant restrictive pore with a radius of approximately 4.5 Angstrom and a much larger but infrequent, non-restrictive pore, were differentially regulated by IFNgamma. Incubation with IFNgamma dose-dependently and reversibly increased the frequency of the non-restrictive pores while having no significant effect on the restrictive component. Cytokine-induced increases in beta, the descriptor of the non-restrictive pore, correlated closely with increased permeability to large molecules (10 kDa) including E. coli-derived lipopolysaccharide, but not small (0.182 kDa) molecules. This effect was associated with changes in expression of the tight junction proteins occludin and claudin-1. These data suggest that IFNgamma selectively increases the transepithelial flux of large molecules by activating specific pathways within the junctional pore. One hypothesis is that this process may be activated in the early stages of the inflammatory response, facilitating the passage of large and potentially antigenic molecules across the gut without gross disruption of the barrier to small molecules.

Caco-2 Cells↗
PubMed2005

Desmosomal cadherin misexpression alters beta-catenin stability and epidermal differentiation.

Desmosomal adhesion is important for the integrity and protective barrier function of the epidermis and is disregulated during carcinogenesis. Strong adhesion between keratinocytes is conferred by the desmosomal cadherins, desmocollin (Dsc) and desmoglein. These constitute two gene families, members of which are differentially expressed in epidermal strata. It has been suggested that this stratum-specific expression regulates keratinocyte differentiation. We tested this hypothesis by misdirecting the expression of the basally abundant desmosomal cadherins Dsc3a and Dsc3b to suprabasal differentiating keratinocytes in transgenic mice. No phenotype was apparent until adulthood, when mice developed variable ventral alopecia and had altered keratinocyte differentiation within affected areas. The follicular changes were reminiscent of changes in transgenic mice with an altered beta-catenin stability. Stabilized beta-catenin and increased beta-catenin transcriptional activity were demonstrated in transgenic mice prior to the phenotypic change and in transgenic keratinocytes as a consequence of transgene expression. Hence, a link between desmosomal cadherins and beta-catenin stability and signaling was demonstrated, and it was shown that desmocollin cadherin expression can affect keratinocyte differentiation. Furthermore, the first function for a "b-type" desmocollin cadherin was demonstrated.

Alopecia↗
PubMed2003

Mast cells disrupt epithelial barrier function during enteric nematode infection.

We have investigated the influence of mast cells on the barrier function of intestinal epithelium during nematode infection. Trichinella spiralis infection induces a strong type 2 cytokine-mediated inflammation, resulting in a critical mucosal mastocytosis that is known to mediate expulsion of the parasites from the intestine. The host response to infection is also characterized by an increase in mucosal leakiness. We show here that intestinal epithelial permeability is markedly elevated during infection, with kinetics that mirror the adaptive immune response to primary and secondary infection. Furthermore, we have identified degradation of the tight junction protein, occludin, thereby providing a mechanism for increased paracellular permeability during helminth infection. We further demonstrate by using anti-c-kit antibody and IL-9 transgenic mice that mast cells are directly responsible for increasing epithelial paracellular permeability and that mice deficient in a mast cell-specific protease fail to increase intestinal permeability and fail to expel their parasite burden. These results provide the mechanism whereby mucosal mast cells mediate parasite expulsion from the intestine.

Animals↗
PubMed2002

Calcium induces differentiation of primary human salivary acinar cells.

Cultivation of human parotid glands in serum-free medium (Ca(2+) concentration, 0.2 mM) with growth supplements resulted in isolation of a homogeneous population of epithelial cells without any mesenchymal cells. The isolated cells showed an undifferentiated phenotype with scant cytoplasmic organelles, and low levels of alpha-amylase expression. The cells remained viable and undifferentiated for up to 24 passages when subcultured at 80% confluence in 0.2 mM Ca(2+) medium with a 1:3 split ratios. There was little cell-cell contact. A Ca(2+) switch from 0.2 to 1 mM induced cell-cell contact with translocation of desmosomal proteins from the cytoplasm to the cell membrane, and sequential differentiation of serous acinar cells with a glandular arrangement, well-developed cytoplasmic organelles and an increased level of alpha-amylase expression. These morphological changes and desmosome assembly were blocked by treatment with non-specific PKC inhibitor. Moreover, the addition of PKC activator, tetradecanoylphorbol 13-acetate (TPA), to 0.2 mM Ca(2+) medium caused transient assembly of desmosome-like structure, but did not induce cell-cell contact or morphological differentiation. Cultivation of the cells in 1.5 mM Ca(2+) medium resulted in increased stratification of the cells and reduced alpha-amylase expression. These findings provide the first demonstration that continuous cultivation in 1.0 mM Ca(2+) medium is required for cellular differentiation of salivary gland acinar cells, and maintenance of the differentiated state.

Blotting, Western↗
PubMed2002

Desmosomal cadherins.

New evidence from blocking desmosomal adhesion with anti-adhesion peptides reveals a role for desmosomes in cell positioning in morphogenesis. Desmosomal adhesion is necessary for the stability of adherens junctions in epithelial cell sheets. Knockout and mis-expression of desmosomal cadherins in mice suggests that they may function directly or indirectly in regulating epidermal differentiation. Protein kinase C signalling and tyrosine phosphorylation appear to regulate desmosomal adhesion. There are new insights into the role of desmosomal cadherins in autoimmune, infectious and genetic disease.

Animals↗
PubMed2002

Suprabasal desmoglein 3 expression in the epidermis of transgenic mice results in hyperproliferation and abnormal differentiation.

The desmoglein 1 (Dsg1) and desmocollin 1 (Dsc1) isoforms of the desmosomal cadherins are expressed in the suprabasal layers of epidermis, whereas Dsg3 and Dsc3 are more strongly expressed basally. This differential expression may have a function in epidermal morphogenesis and/or may regulate the proliferation and differentiation of keratinocytes. To test this hypothesis, we changed the expression pattern by overexpressing human Dsg3 under the control of the keratin 1 (K1) promoter in the suprabasal epidermis of transgenic mice. From around 12 weeks of age, the mice exhibited flaking of the skin accompanied by epidermal pustules and thinning of the hair. Histological analysis of affected areas revealed acanthosis, hypergranulosis, hyperkeratosis, localized parakeratosis, and abnormal hair follicles. This phenotype has some features in common with human ichthyosiform diseases. Electron microscopy revealed a mild epidermal spongiosis. Suprabasally, desmosomes showed incorporation of the exogenous protein by immunogold labeling but were normal in structure. The epidermis was hyperproliferative, and differentiation was abnormal, demonstrated by expression of K14 in the suprabasal layer, restriction of K1, and strong induction of K6 and K16. The changes resembled those found in previous studies in which growth factors, cytokines, and integrins had been overexpressed in epidermis. Thus our data strongly support the view that Dsg3 contributes to the regulation of epidermal differentiation. Our results contrast markedly with those recently obtained by expressing Dsg3 in epidermis under the involucrin promoter. Possible reasons for this difference are considered in this paper.

Animals↗
PubMedDate not supplied

Peptidase allergen Der p 1 initiates apoptosis of epithelial cells independently of tight junction proteolysis.

Loss of epithelial cell polarity, which can arise following disruption of tight junctions (TJs), is a precursor to the care-fully orchestrated removal of moribund cells from epithelia in apoptosis. Ordinarily, this cycle of events has minimally disruptive effects on the function of the epithelial barrier, but some agents have been identified that induce apoptosis and promote epithelial leakiness. The allergen Der p 1 is a cysteine peptidase that cleaves TJ adhesion proteins and induces apoptosis in epithelial cells. This suggests the possibility that, at least for some inducers of apoptosis, these events might be causally linked. We report here that Der p 1 induces epithelial apoptosis before outright cell detachment and that apoptosis occurs within the same time span as increased paracellular permeability in polarized epithelial monolayers. Whilst TJ-deficient BEAS-2B cells were resistant to Der p 1-induced apoptosis, the cell line 1HAEo-, which was also TJ deficient, was sensitive to Der p 1, providing evidence against TJ proteolysis as a cause of apoptosis. To provide direct evidence, we propagated cells that normally express TJs in low calcium medium that prevented intercellular junction assembly. These cells retained full susceptibility to Der p 1, indicating that Der p 1-induced apoptosis is independent from TJ proteolysis.

Allergens↗
PubMedDate not supplied

Desmosomal adhesion: structural basis, molecular mechanism and regulation (Review).

Desmosomes are adhesive intercellular junctions of epithelia and cardiac muscle. They have an essential function in maintaining the integrity of tissues, which is compromised in human genetic and autoimmune disease that targets desmosomal components. Recent evidence (1) suggests new roles for the function of desmosomal adhesion in tissue morphogenesis, (2) gives new insights into the molecular mechanism of adhesion, (3) indicates that the desmosomal adhesion molecules, desmocollin and desmoglein, may contribute to the regulation of epidermal diffentiation, and (4) shows that the affinity of desmosomal adhesion is regulated by protein kinase C.

Animals↗