Transepithelial transport and mucosal defence II: secretion of IgA

In this second article on mucosal defence and transepithelial transport, Jean-Pierre Kraehenbuhl and Marian Neutra discuss the part played by a special class of antibody, polymeric IgA, in the protection of mucosal surfaces lining the digestive, respiratory and genital tracts, and the implications for mucosal vaccines. Polymeric IgA crosslinks luminal antigens or pathogens, thus preventing their interaction with epithelial cells. Following stimulation by antigen in the organized mucosal lymphoid tissue, effector B lymphocytes enter the circulation and migrate to distant mucosal or glandular sites, where they differentiate into polymeric-IgA-producing plasma cells. These antibodies reach the environment by transport across the epithelial cells of mucosal and glandular tissues.     

The role of M cells in the protection of mucosal membranes

 The mucosa-associated lymphoid tissues continuously take up antigenic matter from the lumen to generate immune responses or to maintain immune tolerance. This antigen sampling is performed by highly specialised epithelial cells, the membranous (M) cells of the dome epithelia. M cells possess a unique ultrastructure and lie in close contact to lymphoid cells. They endocytose soluble and solid substances, including entire microorganisms, at their apical membrane, transport these in vesicles to their basolateral membrane and exocytose them to the intercellular space. This review summarises the structural and functional peculiarities of M cells in different species and at the different sites of lymphoid tissue along the digestive and respiratory tracts. Specialisations of M cells for antigen uptake and transport comprise the composition of their apical membrane and its glycocalyx, a modified cytoskeleton as compared to enterocytes and a pocket-like invagination of the basolateral membrane populated by lymphocytes and macrophages. Besides ultrastructural characteristics, histochemical markers are listed that are currently available for detecting M cells by light microscopy. The origin, differentiation and distribution of M cells and other epithelial cell types of the dome epithelium are outlined. As M cells are used as entry sites by various pathogens and, in the future, might be employed for the oral application of vaccines and drugs, the clinical relevance of M cells in health and disease is discussed. Accepted: 4 August 1997     

Cytokine responses during mucosal infections: role in disease pathogenesis and host defence

Mucosal pathogens use diverse and highly specific molecular mechanisms to activate mucosal inflammation. It may even be argued that their virulence depends on the inflammatory response that they induce. Some bacteria target epithelial cells and trigger them to produce inflammatory mediators but others cross the mucosa and activate macrophages or dendritic cells. Although systemic release of inflammatory mediators causes many symptoms and signs of infection, local chemokine production leads to the recruitment of inflammatory cells and lymphocytes that participate directly in the clearance of bacteria from mucosal sites. In this way, mucosal inflammation is a two-edged sword responsible for disease associated tissue destruction and crucial for the antimicrobial defence. Understanding of these pathways should create tools to enhance the defence and interfere with disease.     

Transepithelial transport in cell culture: Bioenergetics of Na-, D-glucose-coupled transport

The renal cell line LLC-PK₁ contransports Na and D -glucose from the apical to the basolateral side of the cell monolayer, and the short-circuit current (Isc) measures the net amount of Na transported. Under conditions of maximal cotransport, the addition of phlorizin or removal of Na rreversibly decreased oxygen consumption by one-hal. In the absence of glycolytic substrates, α-methyl-D -glucoside stimulated Isc and oxygen consumption, although the Isc came to a steady state 50% less than when glycolytic substrates were present. The addition of other aerobic substrates did not increase Isc; however, when non-contransported glycolytic substrates were introduced the Isc returned to a maximum with an associated fall in oxygen consumption and increased lactate production. Thus, in the absence of glycolytic substrates aerobic ATP formation may be rate-limiting for Na, D -glucose contansport. For this epithelium glycolysis makes an impotant contribution to the provision of energy or transport. Oxygen consumption does not correlate well with Isc and is not a good measured off the energy used in transport.     

Transepithelial Na+ transport and the intracellular fluids: A computer study

Summary Computer simulations of tight epithelia under three experimental conditions have been carried out, using the rheogenic nonlinear model of Lew, Ferreira and Moura (Proc. Roy. Soc. London. B 206:53–83, 1979) based largely on the formulation of Koefoed-Johnsen and Ussing (Acta Physiol. Scand.42:298–308, 1958). First, analysis of the transition between the short-circuited and open-circuited states has indicated that (i) apical Cl^– permeability is a critical parameter requiring experimental definition in order to analyze cell volume regulation, and (ii) contrary to certain experimental reports, intracellular Na⁺ concentration (c _Na ^c ) is expected to be a strong function of transepithelial clamping voltage. Second, analysis of the effects of lowering serosal K⁺ concentration (c _K ^s ) indicates that the basic model cannot simulate several well-documented observations; these defects can be overcome, at least qualitatively, by modifying the model to take account of the negative feedback interaction likely to exist between the apical Na⁺ permeability andc _Na ^c . Third, analysis of the effects induced by lowering mucosal Na⁺ concentration (c _Na ^m ) strongly supports the concept that osmotically induced permeability changes in the apical intercellular junctions play a physiological role in conserving the body's stores of NaCl. The analyses also demonstrate that the importance of Na⁺ entry across the basolateral membrane is strongly dependent upon transepithelial potential,c _Na ^m andc _K ^s ; under certain conditions, net Na⁺ entry could be appreciably greater across the basolateral than across the apical membrane.     

Adrenomedullin and mucosal defence: interaction between host and microorganism

Many surface epithelial cells express adrenomedullin (AM) and it is postulated that it may have an important protective role. This peptide has many properties in common with other cationic antimicrobial peptides including the human beta-defensins. Antimicrobial activity against members of the human skin, oral, respiratory tract and gastric microflora has been demonstrated. Both pathogenic and commensal strains of bacteria are sensitive; Gram-positive and Gram-negative bacteria being equally susceptible. No activity against the yeast Candida albicans was observed. Minimum inhibitory and minimum bacteriocidal concentrations range from 7.75 x 10(-4) to 12.5 and 0.003 to >25.0 microg ml(-1), respectively. On exposure of oral, skin and gastric epithelial cells to whole cells and culture supernatants from bacteria isolated from these sites an increase in AM peptide and gene expression has been observed. No upregulation was detected with C. albicans. In cultured cells and an animal infection model increased AM peptide and gene expression has been demonstrated using immunohistochemical and in situ hybridization techniques. These collective findings suggest that AM represents a new category of antimicrobial peptide, which contributes to the mucosal host defence system.     

Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. I. Morphological evidence

The G protein of vesicular stomatitis virus was implanted in the apical plasma membrane of Madin-Darby canine kidney cells by low pH-dependent fusion of the viral envelope with the cellular membrane. The amount of fusion as determined by removal of unfused virions, either by tryptic digestion or by EDTA treatment at 0 degree C, was 22-24% of the cell- bound virus radioactivity. Upon incubation of cells after implantation, the amount of G protein as detected by immunofluorescence diminished on the apical membrane and appeared within 30 min on the basolateral membrane. At the same time some G protein fluorescence was also seen in intracellular vacuoles. The observations by immunofluorescence were confirmed and extended by electron microscopy. Using immunoperoxidase localization, G protein was seen to move into irregularly shaped vacuoles (endosomes) and multivesicular bodies and to appear on the basolateral plasma membrane. These results suggest that the apical and basolateral domains of Madin-Darby canine kidney cells are connected by an intracellular route.     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.     

M细胞在肠黏膜免疫中作用的研究进展

M细胞是肠道一种免疫细胞,同时,也是一种特殊的抗原运转细胞。M细胞具有特殊的形态结构特点,与肠黏膜免疫功能密切相关。目前认为,位于肠淋巴滤泡上皮中特化的M细胞是大多数黏膜病原体侵入机体的靶细胞,它能特异性的结合肠道大分子物质及微生物,并将其摄取、转运至位于其下的APC进行识别、处理,并激活T、B淋巴细胞,继而激发肠道黏膜免疫应答作用。本研究就目前国内外学者所做M细胞在肠黏膜免疫中作用的研究进展做一综述。     

Transepithelial transport and enzymatic detoxification of gluten in gluten-sensitive rhesus macaques

Background and Aims

In a previous report, we characterized a condition of gluten sensitivity in juvenile rhesus macaques that is similar in many respects to the human condition of gluten sensitivity, celiac disease. This animal model of gluten sensitivity may therefore be useful toward studying both the pathogenesis and the treatment of celiac disease. Here, we perform two pilot experiments to demonstrate the potential utility of this model for studying intestinal permeability toward an immunotoxic gluten peptide and pharmacological detoxification of gluten in vivo by an oral enzyme drug candidate.

Methods

Intestinal permeability was investigated in age-matched gluten-sensitive and control macaques by using mass spectrometry to detect and quantify an orally dosed, isotope labeled 33-mer gluten peptide delivered across the intestinal epithelium to the plasma. The protective effect of a therapeutically promising oral protease, EP-B2, was evaluated in a gluten-sensitive macaque by administering a daily gluten challenge with or without EP-B2 supplementation. ELISA-based antibody assays and blinded clinical evaluations of this macaque and of an age-matched control were conducted to assess responses to gluten.

Results

Labeled 33-mer peptide was detected in the plasma of a gluten-sensitive macaque, both in remission and during active disease, but not in the plasma of healthy controls. Administration of EP-B2, but not vehicle, prevented clinical relapse in response to a dietary gluten challenge. Unexpectedly, a marked increase in anti-gliadin (IgG and IgA) and anti-transglutaminase (IgG) antibodies was observed during the EP-B2 treatment phase.

Conclusions

Gluten-sensitive rhesus macaques may be an attractive resource for investigating important aspects of celiac disease, including enhanced intestinal permeability and pharmacology of oral enzyme drug candidates. Orally dosed EP-B2 exerts a protective effect against ingested gluten. Limited data suggest that enhanced permeability of short gluten peptides generated by gastrically active glutenases may trigger an elevated antibody response, but that these antibodies are not necessarily causative of clinical illness.     

The rodlet cells of teleostean fish: their potential role in host defence in relation to the role of mast cells/eosinophilic granule cells

The distribution and potential function of the rodlet cells of teleosts were studied by microscopic observations on tissue samples from the digestive tract and adjacent tissues, including the bulbus arteriosus. Fish representing 3-5 genera from each of the families Salmonidae, Cyprinidae, Gadidae and Labridae were included in the study. Great individual variations in the distribution of rodlet cells were found in all species of salmonids, gadids and labrids. The cells seemed to be absent in some individuals of a species and were associated with different epithelial tissues in others, but were not found in vascular endothelia. Their occurrence was common in all salmonids caught in their natural environment, whereas those in aquaculture, kept under controlled conditions with respect to water quality, showed extremely few rodlet cells. In species of the cyprinid family, the picture was different. Rodlet cells were consistently present under the endothelium of the bulbus arteriosus, and were very numerous at this location in individuals infected with blood flukes. In other epithelial tissues of cyprinids, rodlet cells were encountered in fairly high numbers, but in some tissues of individuals from all species they were occasionally absent. In all of the studied families rodlet cells seemed to be recruited when helminths affected epithelial tissues. Mast cells/eosinophilic granule cells were consistently very numerous in tissues of the intestine of cyprinids and labrids. In gadids, mast cells/eosinophilic granule cells seemed to be absent. Present evidence points to a role for the rodlet cells in defence functions, e.g. in combating helminths, and the suggestion earlier made for mast cells/eosinophilic granule cells, that evolution has created a "standing force" in particular tissues of teleosts consistently exposed to pathogens, whereas an efficient "mobilization force" has been an advantage in those living in more pathogen-free environments, may also be applied to rodlet cells, explaining the differences between teleostean families with respect to their distribution pattern.     

The role of electron transport in the defence response of the South African abalone, Haliotis midae

In order to establish health management systems for farmed abalone, it is necessary to understand how the abalone immune system functions and responds to stimulation. Two electron transport system genes, cytochrome b and cytochrome c oxidase III, were found to be upregulated in a cDNA microarray experiment performed on haemocytes from immune-stimulated abalone (Arendze-Bailey, unpublished). The current study sought to elucidate the role of these genes, and thus the electron transport system, in the abalone immune response by specifically inhibiting cytochrome b with antimycin A and measuring haemocyte immune parameters in vivo. Antimycin A did not decrease haemocyte cell viability, but halved cellular ATP from 4 x 10(12) nM/cell to 2 x 10(12) nM/cell (p < 0.05, unpaired t-test). Inhibition of electron transport resulted in a 0.6 fold increase in cellular superoxide levels (p < 0.05, unpaired t-test), while phagocytosis dropped by nearly 50% (p < 0.05, ANOVA) and the ability of haemocytes to kill bacteria was also reduced. Since cytochrome b and cytochrome c oxidase III expression is upregulated in immune-stimulated abalone, and inhibition of electron transport resulted in a decreased immune response in vivo, we conclude that the abalone immune response is dependent on electron transport and that oxidative phosphorylation plays a role in the immune response following stimulation.     

Transepithelial transport of flavanone in intestinal Caco-2 cell monolayers

Our recent study [S. Kobayashi, S. Tanabe, M. Sugiyama, Y. Konishi, Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers, Biochim. Biophys. Acta, 1778 (2008) 33-41] shows that the mechanism of absorption of hesperetin involves both proton-coupled active transport and transcellular passive diffusion. Here, as well as analyzing the cell permeability of hesperetin, we also study the transport of other flavanones, naringenin and eriodictyol, using Caco-2 cell monolayers. Similar to hesperetin mentioned, naringenin and eriodictyol showed proton-coupled polarized transport in apical-to-basolateral direction in non-saturable manner, constant permeation in the apical-to-basolateral direction (J_ap → bl) irrespective of the transepithelial electrical resistance (TER), and preferable distribution into the basolateral side after apical loading in the presence of a proton gradient. Furthermore, the proton-coupled J_ap → bl of hesperetin, naringenin and eriodictyol, were inhibited by substrates of the monocarboxylic acid transporter (MCT), such as benzoic acid, but not by ferulic acid. In contrast, both benzoic and ferulic acids have no stimulatory effect on J_ap → bl of each flavanone by trans-stimulation analysis. These results indicates that proton-driven active transport is commonly participated in the absorption of flavanone in general, and that its transport is presumed to be unique other than MCT-mediated transport for absorption of phenolic acids (PAs), sodium-dependent MCT (SMCT) nor anion exchanger-mediated transport.