Short-term tissue permeability actions of dextran sulfate sodium studied in a colon organ culture system.

Dextran sulfate sodium (DSS)-induced colitis is the most commonly used animal model for inflammatory bowel diseases. However, the precise molecular action of DSS, in particular its initial effect on the epithelial tissue permeability, is still poorly understood. In the present work, organ culture of mouse - and pig colon explants were performed for 1-2 h in the presence/absence of 2% DSS together with polar- and lipophilic fluorescent probes. Probe permeability was subsequently assessed by fluorescence microscopy. DSS rapidly increased paracellular permeability of 70-kDa dextran without otherwise affecting the overall epithelial integrity. FITC-conjugated DSS likewise permeated the epithelial barrier and strongly accumulated in nuclei of cells scattered in the lamina propria. By immunolabeling, plasma cells, T cells, macrophages, mast cells, and fibroblasts were identified as possible targets for DSS, indicating that accumulation of the polyanion in nuclei was not confined to a particular type of cell in the lamina propria. In contrast, colonocytes were rarely targeted by DSS, but as visualized by transmission electron microscopy, it induced the formation of vacuole-like structures in the intercellular space between adjacent epithelial cells. Nuclei of various cell types in the lamina propria, including both cells of the innate and adaptive immune system, are novel targets for a rapid action of DSS, and from previous in vitro studies, polyanions like DSS are known to disrupt nucleosomes by binding to the histones. We therefore propose that nuclear targeting is one way whereby DSS exerts its inflammatory action as a colitogen in animal models of inflammatory bowel diseases.

Probing paracellular -versus transcellular tissue barrier permeability using a gut mucosal explant culture system.

Intestinal permeation enhancers (PEs), i.e. agents improving oral delivery of therapeutic drugs with poor bioavailability, may typically act by two principally different mechanisms: to increase either transcellular -or paracellular passage across the epithelium. With the aim to define these different modes of action in a small intestinal mucosal explant system, the transcellular-acting PE sodium dodecyl sulfate (SDS) was compared to the paracellular-acting PE ethylenediaminetetraacetic acid (EDTA), using several fluorescent polar - and lipophilic probes. Here, SDS rendered the enterocyte cell membranes leaky for the relatively small polar tracers Lucifer yellow and a 3 kD Texas red-conjugated dextran, but most conspicuously SDS blocked constitutive endocytosis from the brush border. In contrast, the main action of EDTA was to increase paracellular passage across the epithelium of both polar probes, including 10 - and 70 kDa dextrans and lipophilic probes, visualized by distinct stripy lateral staining of enterocytes and/or accumulation in the lamina propria. In addition, EDTA caused a loss of epithelial cell polarity by opening tight junctions for diffusion of domain-specific basolateral/apical cell membrane protein markers into the opposite domains. By transmission electron microscopy, SDS caused the formation of vacuoles and vesicle-like structures at the lateral cell membranes. In contrast, EDTA led to a bulging of the whole enterocyte apex, resulting in a "cobblestone" appearance of the epithelium, probably caused by an extreme contraction of the perijunctional actomyosin ring. We conclude that the mucosal explant system is a convenient model for predicting transcellular/paracellular modes of action of novel prospective PEs.

Probing the Action of Permeation Enhancers Sodium Cholate and N-dodecyl-ß-D-maltoside in a Porcine Jejunal Mucosal Explant System.

The small intestinal epithelium constitutes a major permeability barrier for the oral administration of therapeutic drugs with poor bioavailability, and permeation enhancers (PEs) are required to increase the paracellular and/or transcellular uptake of such drugs. Many PEs act as surfactants by perturbing cell membrane integrity and causing permeabilization by leakage or endocytosis. The aim of the present work was to study the action of sodium cholate (NaC) and N-dodecyl-ß-D-maltoside (DDM), using a small intestinal mucosal explant system. At 2 mM, both NaC and DDM caused leakage into the enterocyte cytosol of the fluorescent probe Lucifer Yellow, but they also blocked the constitutive endocytotic pathway from the brush border. In addition, an increased paracellular passage of 3-kDa Texas Red Dextran into the lamina propria was observed. By electron microscopy, both PEs disrupted the hexagonal organization of microvilli of the brush border and led to the apical extrusion of vesicle-like and amorphous cell debris to the lumen. In conclusion, NaC and DDM acted in a multimodal way to increase the permeability of the jejunal epithelium both by paracellular and transcellular mechanisms. However, endocytosis, commonly thought to be an uptake mechanism that may be stimulated by PEs, was not involved in the transcellular process.

Impact of cell-penetrating peptides (CPPs) melittin and Hiv-1 Tat on the enterocyte brush border using a mucosal explant system.

"Cell penetrating peptides" (CPPs) are natural or synthetic peptides with the ability to interact with cell membranes in order to enter cells and/or deliver cargo. They attract considerable interest as permeation enhancers for oral delivery of therapeutic drugs with poor bioavailability, such as proteins or DNA. A main barrier is the intestinal epithelium where passage needs to proceed through a paracellular -and/or a transcellular pathway. Using an organ cultured mucosal explant model system and a selection of fluorescent polar -and lipophilic tracers, the aim of the present study was to investigate the interaction of two CPPs, melittin and Hiv-1 Tat, with the enterocyte brush border. Melittin belongs to the amphipathic class of CPPs, and within 0.5-1 h it bound to, and penetrated, the enterocyte brush border, causing leakage into the cytosol and increased paracellular passage into the lamina propria. Surprisingly, melittin also abolished endocytosis of tracers from the brush border into early endosomes in the terminal web region (TWEEs), excluding any permeation enhancing effect via such an uptake mechanism. Electron microscopy revealed that melittin caused an elongation of the brush border microvilli and a reduction in their diameter. HIV-1 Tat is a cationic CPP that is internalized by cells due to a sequence, mainly of arginines, from residue 49 to 57, and a peptide containing this sequence permeabilized enterocytes to a polar tracer by a leakage into the cytosol. In conclusion, the CPPs studied acted by causing leakage of tracers into the enterocyte cytosol, not by inducing endocytosis.

Intestinal surfactant permeation enhancers and their interaction with enterocyte cell membranes in a mucosal explant system.

Intestinal permeation enhancers (PEs) are agents aimed to improve oral delivery of therapeutic drugs with poor bioavailability. The main permeability barrier for oral delivery is the intestinal epithelium, and PEs act to increase the paracellular and/or transcellular passage of drugs. Transcellular passage can be achieved by cell membrane permeabilization and/or by endocytic uptake and subsequent transcytosis. One broad class of PEs is surfactants which act by inserting into the cell membrane, thereby perturbing its integrity, but little is known about how the dynamics of the membrane are affected. In the present work, the interaction of the surfactants lauroyl-L-carnitine, 1-decanoyl-rac-glycerol, and nonaethylene glycol monododecyl ether with the intestinal epithelium was studied in organ cultured pig jejunal mucosal explants. As expected, at 2 mM, these agents rapidly permeabilized the enterocytes for the fluorescent polar tracer lucifer yellow, but surprisingly, they all also blocked both constitutive -and receptor-mediated pathways of endocytosis from the brush border, indicating a complete arrest of apical membrane trafficking. At the ultrastructural level, the PEs caused longitudinal fusion of brush border microvilli. Such a membrane fusogenic activity could also explain the observed formation of vesicle-like structures and large vacuoles along the lateral cell membranes of the enterocytes induced by the PEs. We conclude that the surfactant action of the PEs selected in this study not only permeabilized the enterocytes, but profoundly changed the dynamic properties of their constituent cell membranes.

IgG trafficking in the adult pig small intestine: one- or bidirectional transfer across the enterocyte brush border?

Immunoglobulin G (IgG) transfer in opposite directions across the small intestinal brush border serves different purposes in early life and in adulthood. In the neonate, maternal IgG is taken up from the gut lumen into the blood, conferring passive immunity to the offspring, whereas in the adult immunoglobulins, including IgG made by plasma cells in the lamina propria, are secreted via the brush border to the lumen as part of the mucosal defense. Here, IgG has been proposed to perform a luminal immune surveillance which eventually includes a reuptake through the brush border as pathogen-containing immune complexes. In the present work, we studied luminal uptake of FITC-conjugated and gold-conjugated IgG in cultured pig jejunal mucosal explants. After 1 h, binding to the brush border was seen in upper crypts and lower parts of the villi. However, no endocytotic uptake into EEA-1-positive compartments was detected, neither at neutral nor acidic pH, despite an ongoing constitutive endocytosis from the brush border, visualized by the polar tracer CF594. The 40-kDa neonatal Fc receptor, FcRn, was present in the microvillus fraction, but noteworthy, a 37 kDa band, most likely a proteolytic cleavage product, bound IgG in a pH-dependent manner more efficiently than did the full-length FcRn. In conclusion, our work does not support the theory that bidirectional transfer of IgG across the intestinal brush border is part of the luminal immune surveillance in the adult.

Organ Culture as a Model System for Studies on Enterotoxin Interactions with the Intestinal Epithelium.

Studies on bacterial enterotoxin-epithelium interactions require model systems capable of mimicking the events occurring at the molecular and cellular levels during intoxication. In this chapter, we describe organ culture as an often neglected alternative to whole-animal experiments or enterocyte-like cell lines. Like cell culture, organ culture is versatile and suitable for studying rapidly occurring events, such as enterotoxin binding and uptake. In addition, it is advantageous in offering an epithelium with more authentic permeability/barrier properties than any cell line, as well as a subepithelial lamina propria, harboring the immune cells of the gut mucosa.

Small molecule pinocytosis and clathrin-dependent endocytosis at the intestinal brush border: Two separate pathways into the enterocyte.

Pinocytosis at the small intestinal brush border was studied in postweaned porcine cultured mucosal explants, using the fluorescent polar probes Alexa hydrazide (AH, MW 570), Texas red dextran (TRD, MW ~ 3000), and Cascade blue dextran (CBD, MW ~ 10,000). Within 1 h, AH appeared in a string of subapical punctae in enterocytes, indicative of an ongoing constitutive pinocytosis. By comparison, TRD was taken up less efficiently into the same compartment, and no intracellular labeling of CBD was detectable, indicating that only small molecules are pinocytosed from the postweaned gut lumen. AH remained in the terminal web region in EEA-1-positive endosomes ("TWEEs") for at least 2 h, implying that the pinocytic uptake does not proceed towards a transcytic pathway. Like AH, cholera toxin B subunit (CTB) was readily internalized, but the two probes appeared in completely non-overlapping subapical compartments, indicating the existence of two different uptake mechanisms operating simultaneously at the brush border. CTB is internalized by clathrin-dependent receptor mediated endocytosis, but surprisingly the toxin also caused a rapid disappearance from the apical cell surface of two major brush border enzymes, alkaline phosphatase and aminopeptidase N, demonstrating the disruptive effect of this pathway. By immunofluorescence, caveolin-1 was hardly detectable in enterocytes, arguing against a caveolae-mediated uptake of AH, whereas the pinocytosis/phagocytosis inhibitors dimethyl amiloride and cytochalasin D both arrested AH uptake. We propose that the constitutive pinocytic mechanism visualized by AH contributes to maintenance of membrane homeostasis and to enrich the contents of lipid raft constituents at the brush border.

The subcellular localization of GABA transporters and its implication for seizure management.

The ability to modulate the synaptic GABA levels has been demonstrated by using the clinically effective and selective GAT1 inhibitor tiagabine [(R)-N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]nipecotic acid]. N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol (EF1502) which not only inhibits GAT1 like tiagabine but also BGT1 has been shown to modulate extrasynaptic GABA levels. The simultaneous inhibition of synaptic and extrasynaptic GABA transporters using tiagabine and EF1502, respectively has been demonstrated to exert a synergistic anticonvulsant effect in several seizure models in mice. The pharmacological profile of these and similar compounds has been thoroughly investigated in in vitro systems, comparing the GAT subtype selectivity with the ability to inhibit GABA uptake in primary cultures of neurons and astrocytes. However, an exact explanation has not yet been found. In the present study, the ability of GATs to form homo and/or heterodimers was investigated as well as to which membrane micro environment the GATs reside. To investigate dimerization of GATs, fusion proteins of GATs tagged with either yellow fluorescent protein or cerulean fluorescent protein were made and fluorescence resonance energy transfer (FRET) was measured. It was found that GATs form both homo- and hetero-dimers in N2A and HEK-293 cells. Microdomain localization of GATs as investigated by detergent resistant membrane fractions after treatment of tissue with Brij-98 or Triton X-100 revealed that BGT1 and GAT1 mostly localize to non-membrane rafts independent of the detergent used. However, GAT3 localizes to membrane rafts when using Brij-98. Taken together, these results suggest that the observed hetero dimerization of GATs in the FRET study is unlikely to have functional implications since the GATs are located to very different cellular compartments and cell types.

Okadaic acid: a rapid inducer of lamellar bodies in small intestinal enterocytes.

Okadaic acid (OA) is a polyether fatty acid produced by marine dinoflagellates and the causative agent of diarrhetic shellfish poisoning. The effect of OA on apical endocytosis in the small intestine was studied in organ cultured porcine mucosal explants. Within 0.5-1 h of culture, the toxin caused hyper protein phosphorylation, but no detectable loss of cell polarity or cytoskeletal integrity of the enterocytes. Using a fluorescent membrane marker, FM dye, endocytosis from the brush border was affected by the toxin. Although constitutive uptake into subapical terminal web-localized early endosomes (TWEEs) occurred unimpeded in the presence of OA, FM condensed in larger subapical structures by 1 h, implying a perturbed endosomal trafficking/maturation. The fluorescent lysosomotropic agent Lysotracker revealed induction of large lysosomal structures by OA. Endocytosis from the brush border was studied at the electron microscopic level using the membrane-impermeable marker Ruthenium Red (RR). Like FM dye, RR was taken up into TWEEs and multivesicular bodies (MVBs). However, OA induced the formation of a large number of lamellar bodies (LBs), a type of lysosome-related organelles. LBs are the hallmark of phospholipidosis, a pathological condition characterized by lysosomal phospholipid accumulation. Phospholipidosis is observed in acquired lysosomal storage diseases and is induced by a large number of cationic amphiphilic drugs. Unlike the latter, however, OA does not act by accumulating in acidic organelles, implying a different toxic mechanism of action. We propose that rapid induction of LBs, an indicator of phospholipidosis, should be included in the future toxicity profile of OA.

Generation of stable lipid raft microdomains in the enterocyte brush border by selective endocytic removal of non-raft membrane.

The small intestinal brush border has an unusually high proportion of glycolipids which promote the formation of lipid raft microdomains, stabilized by various cross-linking lectins. This unique membrane organization acts to provide physical and chemical stability to the membrane that faces multiple deleterious agents present in the gut lumen, such as bile salts, digestive enzymes of the pancreas, and a plethora of pathogens. In the present work, we studied the constitutive endocytosis from the brush border of cultured jejunal explants of the pig, and the results indicate that this process functions to enrich the contents of lipid raft components in the brush border. The lipophilic fluorescent marker FM, taken up into early endosomes in the terminal web region (TWEEs), was absent from detergent resistant membranes (DRMs), implying an association with non-raft membrane. Furthermore, neither major lipid raft-associated brush border enzymes nor glycolipids were detected by immunofluorescence microscopy in subapical punctae resembling TWEEs. Finally, two model raft lipids, BODIPY-lactosylceramide and BODIPY-GM1, were not endocytosed except when cholera toxin subunit B (CTB) was present. In conclusion, we propose that constitutive, selective endocytic removal of non-raft membrane acts as a sorting mechanism to enrich the brush border contents of lipid raft components, such as glycolipids and the major digestive enzymes. This sorting may be energetically driven by changes in membrane curvature when molecules move from a microvillar surface to an endocytic invagination.

Permeabilization of enterocytes induced by absorption of dietary fat.

Absorption of dietary fat in the small intestine involves epithelial exposure to potentially harmful molecules such as bile salts and free fatty acids. We used organ culture of porcine jejunal explants incubated with a pre-digested mixture of fat (plant oil), bile and pancreatin to mimick the physiological process of dietary fat absorption, and short exposures to the fat mixture caused fat droplet accumulation within villus enterocytes. Lucifer yellow (LY), a fluorescent membrane-impermeable polar tracer was included to monitor epithelial integrity. Both in controls and during fat absorption LY penetrated the epithelium and accumulated in the basal lamina and the lamina propria. LY was also seen in the paracellular space, whereas villus enterocytes were generally only weakly labeled except for small amounts taken up by apical endocytosis. In the crypts, however, fat absorption induced cell permeabilization with LY accumulating in the cytosol and nucleus. Morphologically, both apical and basolateral membranes appeared intact, indicating that the leakiness was caused by minor lesions in the membrane. Albeit to a lesser extent, bile alone was capable of permeabilizing crypt cells, implying that the surfactant properties of bile salts are involved in the process. In addition to LY, crypt enterocytes also became permeable for albumin, ovalbumin and insulin. In conclusion, during fat absorption the permeability of the gut epithelium is increased mainly in the crypts. A possible explanation is that cell membranes of immature crypt cells, lacking detergent-resistant lipid raft microdomains, are less resistant to the deleterious effects of bile salts and free fatty acids.

Staphylococcus aureus enterotoxins A- and B: binding to the enterocyte brush border and uptake by perturbation of the apical endocytic membrane traffic.

Enterotoxins of Staphylococcus aureus are among the most common causes of food poisoning. Acting as superantigens they intoxicate the organism by causing a massive uncontrolled T cell activation that ultimately may lead to toxic shock and death. In contrast to our detailed knowledge regarding their interaction with the immune system, little is known about how they penetrate the epithelial barrier to gain access to their targets. We therefore studied the uptake of two staphylococcal enterotoxins (SEs), SEA and SEB, using organ cultured porcine jejunal explants as model system. Attachment of both toxins to the villus surface was scarce and patchy compared with that of cholera toxin B (CTB). SEA and SEB also bound to microvillus membrane vesicles in vitro, but less efficiently than CTB, and the binding was sensitive to treatment with endoglycoceramidase II, indicating that a glycolipid, possibly digalactosylceramide, acts as cell surface receptor at the brush border. Both SEs partitioned poorly with detergent resistant membranes (DRMs) of the microvillus, suggesting a weak association with lipid raft microdomains. Where attachment occurred, cellular uptake of SEA and SEB was also observed. In enterocytes, constitutive apical endocytosis normally proceeds only to subapical early endosomes present in the actomyosin-rich "terminal web" region. But, like CTB, both SEA and SEB penetrated deep into the cytoplasm. In conclusion, the data show that after binding to the enterocyte brush border SEA and SEB perturb the apical membrane trafficking, enabling them to engage in transcytosis to reach their target cells in the subepithelial lamina propria.

Apolipoprotein A-1 (apoA-1) deposition in, and release from, the enterocyte brush border: a possible role in transintestinal cholesterol efflux (TICE)?

Transintestinal cholesterol efflux (TICE) has been proposed to represent a non-hepatobiliary route of cholesterol secretion directly "from blood to gut" and to play a physiologically significant role in excretion of neutral sterols, but so far little is known about the proteins involved in the process. We have previously observed that apolipoprotein A-1 (apoA-1) synthesized by enterocytes of the small intestine is mainly secreted apically into the gut lumen during fasting where its assembly into chylomicrons and basolateral discharge is at a minimal level. In the present work we showed, both by immunomicroscopy and subcellular fractionation, that a fraction of the apically secreted apoA-1 in porcine small intestine was not released from the cell surface but instead deposited in the brush border. Cholesterol was detected in immunoisolated microvillar apoA-1, and it was partially associated with detergent resistant membranes (DRMs), indicative of localization in lipid raft microdomains. The apolipoprotein was not readily released from microvillar vesicles by high salt or by incubation with phosphatidylcholine-specific phospholipase C or trypsin, indicating a relatively firm attachment to the membrane bilayer. However, whole bile or taurocholate efficiently released apoA-1 at low concentrations that did not solubilize the transmembrane microvillar protein aminopeptidase N. Based on these findings and the well known role played by apoA-1 in extrahepatic cellular cholesterol removal and reverse cholesterol transport (RCT), we propose that brush border-deposited apoA-1 in the small intestine acts in TICE by mediating cholesterol efflux into the gut lumen.

Crucial role of alkaline sphingomyelinase in sphingomyelin digestion: a study on enzyme knockout mice.

Alkaline sphingomyelinase (alk-SMase) hydrolyses sphingomyelin (SM) to ceramide in the gut. To evaluate the physiological importance of the enzyme, we generated alk-SMase knockout (KO) mice by the Cre-recombinase-Locus of X-over P1(Cre-LoxP) system and studied SM digestion. Both wild-type (WT) and KO mice were fed ³H-palmitic acid labeled SM together with milk SM by gavage. The lipids in intestinal content, intestinal tissues, serum, and liver were analyzed by TLC. In KO mice, nondigested ³H-SM in the intestinal content increased by 6-fold and the formation of ³H-ceramide decreased markedly, resulting in 98% reduction of ³H-ceramide/³H-SM ratio 1 h after gavage. The absorbed ³H-palmitic acid portion was decreased by 95%. After 3 h, a small increase in ³H-ceramide was identified in distal intestine in KO mice. In feces, ³H-SM was increased by 243% and ceramide decreased by 74% in the KO mice. The KO mice also showed significantly decreased radioactivity in liver and serum. Furthermore, alkaline phosphatase activity in the mucosa was reduced by 50% and histological comparison of two female littermates preliminarily suggested mucosal hypertrophy in KO mice. This study provides definite proof for crucial roles of alk-SMase in SM digestion and points to possible roles in regulating mucosal growth and alkaline phosphatase function.

Dietary free fatty acids form alkaline phosphatase-enriched microdomains in the intestinal brush border membrane.

Free fatty acids released during intralumenal digestion of dietary fat must pass through the enterocyte brush border membrane before triacylglycerol reassembly and subsequent chylomicron delivery to the lymph system. In the present work fluorescent BODIPY fatty acid analogs were used to study this membrane passage in organ cultured intestinal mucosal explants. We found that in addition to a rapid uptake into the cytoplasm, a fraction of the fatty acid analogs were inserted directly into the brush border membrane. Furthermore, a brief exposure of microvillar membrane vesicles to a fat mixture mimicking a physiological solution of dietary mixed micelles, rearranged the lipid raft microdomain organization of the membranes. Thus, the fat mixture generated a low-density subpopulation of microvillar detergent resistant membranes (DRMs) highly enriched in alkaline phosphatase (AP). Since this GPI-linked enzyme is the membrane protein in the brush border with the highest affinity for lipid rafts, this implies that free fatty acids selectively insert stably into these membrane microdomains. We have previously shown that absorption of dietary lipids transiently induce a selective endocytosis of AP from the brush border, and from work by others it is known that fat absorption is accompanied by a rise in serum AP and secretion of surfactant-like particles from enterocytes. We propose that these physiological processes may be triggered by the sequestering of dietary free fatty acids in lipid raft microdomains of the brush border.

Dietary cholesterol induces trafficking of intestinal Niemann-Pick Type C1 Like 1 from the brush border to endosomes.

The transmembrane protein Niemann-Pick C1 Like 1 (NPC1L1) belongs to the Niemann-Pick C1 (NPC1) family of cholesterol transporters and is mainly expressed in the liver and the small intestine. NPC1L1 is believed to be the main transporter responsible for the absorption of dietary cholesterol. Like NPC1, NPC1L1 contains a sterol sensing domain, suggesting that it might be sensitive to dietary cholesterol. To test this hypothesis, mucosal explants were cultured in the presence or absence of cholesterol. In the absence of cholesterol NPC1L1 was localized mainly in the brush border of the enterocyte, colocalizing with the brush border enzyme aminopeptidase N (APN), and only a minor part was present in intracellular compartments. In contrast, following culture in the presence of cholesterol a major part of NPC1L1 was found in intracellular compartments positive for the early endosomal marker early endosome antigen 1, whereas only a minor fraction was left in the brush border. Neither APN, lactase, nor sucrase-isomaltase was endocytosed in parallel, demonstrating that this is a selective cholesterol-induced endocytosis of NPC1L1. Conceivably either the induced internalization could be due to NPC1L1 acting as an endocytic cholesterol receptor or it could be a mechanism to reduce the cholesterol uptake. The fluorescent cholesterol analog NBD-cholesterol readily labeled the cytoplasm also under conditions nonpermissible for endocytosis, arguing against a receptor-mediated uptake. We therefore propose that cholesterol is absorbed by NPC1L1 acting as a membrane transporter and that NPC1L1 is internalized to an endosomal compartment to reduce the absorption of cholesterol.

Lactoferrin targets T cells in the small intestine.

BACKGROUND: Lactoferrin (Lf) belongs to the transferrin family of non-heme iron-binding proteins and is found in milk and mucosal secretions. Consequently, it is now considered a multifunctional protein mainly involved in both the innate and adaptive immune defenses of the organism against various pathogens, and Lf receptors have been identified at the surfaces of a number of different cells. In the small intestine Lf binds to the luminal surface, but its further interaction with the epithelial cells is controversial. METHODS: In the present work, we studied the uptake of Lf in cultured mucosal explants of pig small intestine by immunofluorescence and immunogold microscopy. RESULTS: Lf rapidly bound to the brush border and subsequently appeared in punctae in the apical cytoplasm, indicating internalization into an endosomal compartment. Essentially, no labeling was detected elsewhere in the enterocytes by 2 h incubation. However, in addition to enterocytes, a distinct subpopulation of cells in the lamina propria also took up Lf, most likely from the serosal side of the explants. None of these cells were apoptotic, nor did they belong to the predominant group of immunoglobulin-synthesizing plasma cells in the lamina propria. However, they were CD3(+), identifying them as T lymphocytes. Lf labeling of these cells was mainly seen in the cytosol, but occasionally nuclear staining was seen as well, suggesting a direct regulatory role of Lf. CONCLUSION: We propose that Lf functions in the immune defense of the small intestinal mucosa by targeting the population of T cells in the lamina propria.

Domains of increased thickness in microvillar membranes of the small intestinal enterocyte.

The apical surface of the enterocyte is sculpted into a dense array of cylindrical microvillar protrusions by supporting actin filaments. Membrane microdomains (rafts) enriched in cholesterol and glycosphingolipids comprise roughly 50% of the microvillar membrane and play a vital role in orchestrating absorptive/digestive action of dietary nutrients at this important cellular interface. Increased membrane thickness is believed to be a morphological characteristic of rafts. Thus, we investigated whether the high contents of lipid rafts in the microvillar membrane is reflected in local variations in membrane thickness. We measured membrane thickness directly from electron micrographs of sections of fixed mucosal tissue. Indeed, mapping of the microvillar membrane revealed a biphasic distribution of membrane thickness. As a point of reference the thickness distribution of the basolateral membrane was clearly monophasic. The encountered domains of increased thickness (DITs) occupied 48% of the microvillar membrane and from the data we estimated the area of a single DIT to have a lower limit of 600 nm(2). In other experiments we mapped the organization of biochemically defined lipid rafts by immunogold labeling of alkaline phosphatase, a well documented raft marker. Strikingly, the alkaline phosphatase localized to distinct regions of the membrane in a pattern similar to the observed distribution of DITs. Although we were unable to measure membrane thickness directly on the immunogold labeled specimens, and thereby establish an unequivocal connection between DITs and rafts, we conclude that the brush border membrane of the enterocyte contains microdomains distinguishable both by membrane morphology and protein composition.

Galectin-2 at the enterocyte brush border of the small intestine.

The brush border of pig small intestine is a local hotspot for beta-galactoside-recognizing lectins, as evidenced by its prominent labeling with fluorescent lectin PNA. Previously, galectins 3-4, intelectin, and lectin-like anti-glycosyl antibodies have been localized to this important body boundary. Together with the membrane glycolipids these lectins form stable lipid raft microdomains that also harbour several of the major digestive microvillar enzymes. In the present work, we identified a lactose-sensitive 14-kDa protein enriched in a microvillar detergent resistant fraction as galectin-2. Its release from closed, right-side-out microvillar membrane vesicles shows that at least some of the galectin-2 resides at the lumenal surface of the brush border, indicating that it plays a role in the organization/stabilization of the lipid raft domains. Galectin-2 was released more effectively from the membrane by lactose than was galectin-4, and surprisingly, it was also released by the noncanonical disaccharides sucrose and maltose. Furthermore, unlike galectin-4, galectin-2 was preferentially co-immunoisolated with sucrase-isomaltase rather than with aminopeptidase N. Together, these results show that the galectins are not simply redundant proteins competing for the same ligands but rather act in concert to ensure an optimal cross-linking of membrane glycolipids and glycoproteins. In this way, they offer a maximal protection of the brush border against exposure to bile, pancreatic enzymes and pathogens.