Epithelial cell layer and glycocalyx

The IEC and the monolayer they form play a particularly important role in the intestinal functions. This is the first cell population that comes into direct contact with lumen contents, digest and intestinal microbial components and the final cell layer for absorptive or secretory processes in the epithelium [66, 168, 169]. The S-layer (single cell thick monolayer) is capable of regulating the permeability [170] by expression of a variety of digestive enzymes, transporters and receptors.

The IEC layer is composed of distinct cell lineages, each contributing uniquely to mucosal defense and maintenance of barrier integrity [171-174]. To enable a barrier, polarised IEC are sealed by intercellular junctions to their adjacent cells at the most apical region and bound to the extracellular matrix via several multi-protein complexes [24, 152, 169, 175]. Further, the cells show distinct polarisation by site-specific expression of transport proteins which allows

21

vectorial absorption and secretion [168, 176]. All mature cells of the absorptive, goblet and enteroendocrine cell lines are evolved from the lower crypt compartments consisting of stem cells [164, 177]. During their migration towards the villus, these cells proliferate, turn into transit amplifying cells and finally differentiate into the different lineages. They migrate within one to four days along the vertical axis of the functional villus where they are segregated into the mucus layer [24, 57, 156, 178-180], generating a fast and constant cell turnover [152]. Thereby a balance between proliferation and cell loss is maintained while preserving the structural continuity and functional integrity of the IEC surface [181]. Evidence has been found that apoptosis is activated specifically in villus cells residing at the top of the villi [182]. The most abundant cell types among IEC are enterocytes and goblet cells [183]:

♦ Absorptive enterocytes with a luminal brush-border [57, 184] are the most prevalent cells, they form a layer of columnar-shaped cells or microvillus epithelium to separate the lumen from the sub-epithelial lamina propria or basolateral domain in which the mucosal immune cells reside [173, 185].

♦ Goblet cells are unicellular glands which secret mucins as lubricant for protection of IEC and to bind pathogens [57, 171, 186]. They comprise between 4% and 16% of the cells on the villi [95, 164].

Less abundant cells include paneth cells (antimicrobial defence cells in the small intestine [57, 164, 187, 188]), microfold cells without glycocalyx or mucin layer [97, 189] (transepithelial vesicular trafficking of antigens and bacteria [190, 191]), Peyer’s patches (tertiary lymphoid tissue [78]), chemosensory enteroendocrine cells [168] and the undifferentiated crypt cells [184].

The glycocalyx is a dense extracellular zone (0.5 µm thickness [192]) on the apical surface of IEC which is composed of carbohydrate-rich transmembrane and secreted and vesicular bound molecules (e.g. glycoproteins, glycolipids, proteoglycans or collagen) [193-195]. Because of

22

Figure 1.5: Intestinal layers [196]

Intact intestinal layer system: Lumen with bacteria on the left / bottom part of the picture and IEC in the top-right corner in blue. The two domains are separated by a mucin coat (black diagonal zone) which shows only few bacteria (red) in the outer layer. Colours enhanced to increase contrast.

Figure 1.6: Scheme of the human intestinal layers

Lumen

Bacteria / bacterial biofilm Mucus layer

Epithelial S-layer with glycocalyx Absorptive enterocytes

23

densly glycosylated transmembrane mucins [197] and a relatively fast extracellular release of polysaccharides and proteins from the cell surfaces [198] the cell coat is constantly renewed and acts as reinforcement of the physical barrier. It shows an affinity for lectins, toxins and bacteria [199] and is capped by and integrated within a secreted mucus gel [43, 78, 200-204]. The glycocalyx is thought to play a role in cell growth, differentiation, metabolism and cell-cell interactions [205-210], modulation of receptor-mediated membrane processing [211, 212] and acts as selective molecular sieve and depot region for nutrients [213]. Its diversity and density of saccharides make it an appealing site for lectin-bearing bacteria [24, 214-216].

Levine et al. [213] showed that the total average diffusion times of molecules are increased, and reaction rates decreased, due the anisotropic (density gradient) structure of the extracellular matrix and glycocalyx when compared with isotropic (constant density) transfer media.

1.2.1.1 Cell culture of epithelial cells

To date there have been no reports on the successful culture of freshly dissociated IEC. The most likely explanation lies in the intrinsic properties of the villus enterocytes: they are terminally differentiated cells, destined to undergo apoptosis [182, 217]. Further, the small intestine is one of the most uncommon sites for cancer in humans and experimental animals, and the few cell lines derived from this tissue proved of little use, due to their fibroblastic nature (ovine) [218], slow multiplication rates (10 days) or oncogenic metabolic characteristics (human) [219]. Most well-established tumour derived cell lines originate from colon cancers (e.g. Caco-2 and HT29) [164]. Both Caco-2 and HT29 cells display a marked heterogeneity in morphology and function [220]. Nevertheless, using established cell lines has the advantage that the cells are available and easy to handle, whereas primary cells normally are derived from different individuals and thus may lead to poor reproducibility [55]. However, one should keep in mind that established cell lines are derived from a progressive oncogenic progress and exhibit many chromosomal abnormalities. Further, the cells are from the colon and cannot be assumed to reliably display the properties of normal enterocytes. This demands careful analysis of each

24

individual application, particularly with respect to making meaningful in vitro-in vivo

correlations [55, 164].

1.2.1.2 HT29-MTX cells

The parental cell line HT29 is an isolate of a colon tumour (adenocarcinoma) of a 44-year old Caucasian female [55]. Contrary to earlier assumptions, that the parental HT29 cell line has properties of intestinal stem cells [221], more recent research shows that HT29 cells contain a small proportion of cells which differentiate spontaneously as either enterocytic or mucus secreting cell types. These cells can be selected by special culture conditions, such as applying the cytotoxic drug methotrexate (MTX), deriving a population of clones which maintain a different (mucus secreting) phenotype in the absence of the selective agent [222]. Through stably adapting HT29 cells to MTX, HT29-MTX cells have been generated [221, 222]. Monolayers, grown from sub-clones of HT29-MTX, consist mainly of mature goblet cells with a discrete brush border with the presence of villi and characteristic proteins and form a continuous mucus layer on their apical surface. The thickness of the mucus layer is between 142±51 μm and 53±52 μm, consistent with the range of the human mucus layer [29, 221-223]. Mucin production is dependent on growth phase and starts with confluence and increases post- confluently [224, 225]. At late confluence, HT29-MTX cells show a dense mucus gel with numerous mucus buds on their apical surface [226]. Differentiated HT29-MTX cells secrete several types of mucin, including MUC5AC and MUC6 which are highly expressed in the stomach and upper small intestine [225, 227]. Caution is required, however, as the levels of MUC2, MUC3, MUC4 and MUC5AC mRNA were found to differ from one population to another and within each population according to the confluence stage [225].

Compared to the human small intestine, which secrets mainly MUC2, HT29-MTX cells predominantly produce MUC5AC. Both are secreted sialomucins [228, 229], encoded by a gene cluster on the 11p15 chromosome [230-232]. They can form networks due to cysteine rich N- and C-termini with a minimum of one large proline, threonine and serine rich region (PTS- domain). O-linked glycoside side chains make up <70% of the molecular weight [233, 234].

25

Consensus tandem repeat apo-mucin sequences of the two glycoproteins are PTTTPISTTTTVTPTPTPTGTQT (MUC2) and TTSTTSAP (MUC5AC) [235]. However, differences in the protein structure are of minor importance as interactions are most likely to happen with the easier accessible glycoside side chains which are modified due to malignancy [236, 237]. This manifests in an increase in sialomucin, a decrease in O-acetylated sialic acid (NeuNAc) and sulfomucin or alterations in the expression of blood group antigens [235, 238]. Also a reduction of the molecular weight (due to an approximate 50% reduction in carbohydrate content and chain length), simplification of glycoside chains and aberrant glycosylation in (pre) neoplastic mucins can be observed [235, 237]. The changes which have been described in colon carcinoma cells in vivo, have also been found in mucin-secreting colon carcinoma cells in

culture. For example, mucins of the HT29 cell line have truncated glycoside-side chains due to prematurely stopped elongation due to sialation [239]. Malignant transformation of IEC is also associated with abnormal glycosylation of the cell surface (e.g. gp190 [240] or α2,6-sialation [241]). Despite these variations, HT29 cells and their mucin producing subpopulations are a widely used model for the human intestinal mucin producing cells.

1.2.1.3 Caco-2 cells

Caco-2 cells are the most widely used commercially available cell line (ATCC and ECACC). They were isolated from the colon adenocarcinoma of a 72-year old Caucasian male and express functions of enterocytes upon reaching confluence [242-246]. The cells form a polarised monolayer of well differentiated columnar absorptive cells [164, 247-249]. Thus they are particularly useful for the study of absorption [247, 250], cell polarisation and biosynthesis of brush border enzymes [246, 251-253]. The brush borders (microvilli covered apical cell surface) can be well organised on some cells but quite irregular on others [220]. In addition to brush border enzymes, Caco-2 cells express other features like tight junction proteins [254], the P- glycoprotein drug efflux pump and the di/tripeptide transporter [255-257] which are found in the small intestine.

26

Caco-2 cells have glycosaminoglycans as receptors which can be used for adhesion by microorganisms, e.g. E. coli. As enterococci adherence can be inhibited by treatment with

heparin or heparan sulfate [215, 258], polysaccharides seem to be involved. Similar results are found for eukaryotic cells. Quaroni [259] found that monoclonal antibodies raised against Caco- 2 cells recognised antigens specific to normal villus cells of human colon tissue. However, a Caco-2 monolayer, composed of solely absorptive cells, cannot resemble the small intestinal S- layer by itself. The tightness of the monolayer resembles more the colon [164, 247, 260], about 5- to 10-fold higher transepithelial electric resistance than in the small intestine, resulting in poor permeability for hydrophilic compounds via the paracellular pathway. Furthermore, it is widely accepted that Caco-2 cells show other colonocytic features like the expression of surfactant protein A [261].