Inducible expression of claudin-1-myc but not occludin-VSV-G results in aberrant tight junction strand formation in MDCK cells.

Occludin and 18 distinct members of the claudin family are tetra-span transmembrane proteins that are localized in cell-specific tight junctions (TJs). A previous study showed that expression of chick occludin in Madin-Darby canine kidney (MDCK) cells raised transepithelial electrical resistance (TER) and, paradoxically, increased mannitol flux. In the present study, we employed epitope tagged canine occludin expression, under the control of the tetracycline repressible transactivator, to determine the extent to which the unexpected parallel increase in TER and mannitol flux was related to a structural mismatch between avian and canine occludins, which are only 50% identical. To determine whether the paradoxical changes in permeability was specific to occludin, we assessed the effect of over-expressing epitope tagged murine claudin-1. Our data revealed that over-expression of either of the epitope tagged mammalian tight junction proteins increased TER, mannitol and FITC-dextran flux. We observed a 2- and up to 5.6-fold over-expression of occludin-VSV-G and claudin-1-myc, respectively, with no change in ZO-1, endogenous occludin or claudin-1 expression. Confocal microscopy revealed that occludin-VSV-G, claudin-1-myc and ZO-1 co-localized at the TJ. In addition, claudin-1-myc formed aberrant strands along the lateral cell surface without an underlying ZO-1 scaffold. In fracture labeled replicas these strands consisted of claudin-1-myc with little accompanying occludin. These observations suggest that in epithelial cells claudin-1 can assemble into TJ strands without the participation of either ZO-1 or occludin. The proximity of the myc tag to the COOH-terminal YV sequence of claudin-1 appeared to interfere with its interaction with ZO-1, since over-expression of non-tagged claudin-1 increased TER but had a minimal effect on solute flux and no aberrant strands formed. From our data we conclude that differences in structure between avian and mammalian occludin do not account for the observed paradoxical increase in mannitol flux. Levels of ZO-1 remained unchanged despite substantial increases in induced TJ integral protein expression, suggesting that an imbalance between levels of ZO-1 and occludin or claudin-1 leads to altered regulation of pores through which non-charged solute flux occurs. We suggest that ion and solute flux are differentially regulated at the TJ.

Rapid reduction of MDCK cell cholesterol by methyl-beta-cyclodextrin alters steady state transepithelial electrical resistance.

The role of plasma membrane lipids in regulating the passage of ions and other solutes through the paracellular pathway remains controversial. In this study we explore the contribution of cholesterol (CH) in maintaining the barrier function of an epithelial cell line using the CH-solubilizing agent methyl beta-cyclodextrin (MBCD) to stimulate CH efflux. Inclusion of 20 mM MBCD in both apical and basolateral media reduced CH levels by 70-80% with no significant effect on cell viability. Most of that decrease occurred during the first 30 min of incubation. Recovery of CH content to initial values was nearly complete 22 h after removal of MBCD. Within 30 min of adding MBCD to the culture medium, transepithelial electrical resistance (TER) increased, reaching maximum values 30-40% above controls. This early rise in TER occurred when MBCD was added to either side of the monolayer. The later rapid decline in TER was observed only when MBCD bathed the basolateral surface from which, coincidentally, CH efflux was most rapid. Freeze fracture replicas and transmission electron microscopy of monolayers exposed to MBCD for only 30 min revealed no increase in either the average tight junction (TJ) strand number or the dimensions of the lateral intercellular space. There was a statistically significant increase in the number of TJ particles associated with the E fracture face at this time. This raises the interesting possibility that during CH efflux there is a change in the interaction between TJ particles and underlying cytoskeletal elements. There was no change in staining for occludin and ZO-1. After exposing the basolateral surface to MBCD for 2 h, TER fell below control levels. The accompanying increase in mannitol flux suggests strongly that the decrease in TER resulted from an increase in the permeability of the paracellular and not the transcellular pathway. A decrease in immuno-staining for occludin and ZO-1 at TJs, a striking accumulation of actin at tri-cellular areas as well as a decline in the number of parallel strands, as seen in freeze fracture replicas, suggest that changes in cytoskeletal organization during long incubations with MBCD had physically disrupted the TJ network. Data are presented which suggest that the observed changes in paracellular permeability during CH efflux may be related to increased levels of lipid-derived second messengers, some of which may trigger changes in the phosphorylation status of TJ proteins.

Occludin is a functional component of the tight junction.

Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

Alterations in cell cholesterol content modulate Ca(2+)-induced tight junction assembly by MDCK cells.

Transepithelial electrical resistance (TER), a measure of tight junction (TJ) barrier function, develops more rapidly and reaches higher values after preincubation of MDCK cells for 24 h with 2 microM Lovastatin (lova), an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. While this effect was attributed to a 30% fall in cholesterol (CH), possible effects of lova on the supply of prenyl group precursors could not be excluded. In the current study, strategies were devised to examine effects on TER of agents that simultaneously lower CH and increase the flux of intermediates through the CH biosynthetic pathway. Zaragozic acid, 20 microM, an inhibitor of squalene synthase known to increase the synthesis of isoprenoids and levels of prenylated proteins, lowered cell CH by 30% after 24 h, while accelerating development of TER in the same manner as lova. TER was also enhanced, despite a 23% increase in the rate of [3H]acetate incorporation into CH, when total CH was reduced by 45% during a 2-h incubation with 2 mM methyl beta-cyclodextrin (MBCD), an agent that stimulates CH efflux from cells. The fact that the rate of TER development was diminished when cell CH content was elevated by incubation with a complex of CH and MBCD is further evidence that this sterol modulates development of the epithelial barrier. Cell associated CH derived from the complex was similar to endogenous CH with respect to its accessibility to cholesterol oxidase. Lova's effect on TER was diminished when 5 micrograms/mL of CH was added to the medium during the last 11 h of incubation with lova.

Basolateral but not apical application of protease results in a rapid rise of transepithelial electrical resistance and formation of aberrant tight junction strands in MDCK cells.

In the presence of Ca2+, application of trypsin to the basolateral surface of confluent MDCK cell monolayers with formed tight junctions (TJ), induces the formation of basolaterally oriented aberrant TJ strands. Induction of aberrant TJ strands is accompanied by an increase in transepithelial electrical resistance (TER), up to 90%, which upon addition of trypsin inhibitor is maintained for up to 1 h. Thereafter TER returns slowly to baseline values. Under similar conditions, application of trypsin to the apical surface has little or no effect on either TER or the number of aberrant TJ strands. Confocal microscopy of monolayers, immunostained for ZO-1, revealed that this TJ associated cytoplasmic protein, extended below the TJ along the basolateral surface following brief exposure to trypsin. Removing Ca2+ after treatment of the monolayer with basolaterally applied trypsin resulted, after 20 min, in the increased partitioning of TJ particles onto the E fracture face, of both normal and aberrant TJ strands. Like the TJ strands themselves, therefore, aberrant strands may be linked to cytoskeletal elements. Aberrant TJ strands do not form when monolayers, maintained in low Ca2+ medium, are exposed to trypsin, suggesting that under these conditions TJ precursors, and/or trypsin-sensitive proteins regulating TJ strand assembly, are sequestered in a vesicular compartment that is inaccessible to exogenous trypsin. Prolonged exposure of the apical surface of an established, polarized epithelium with intact TJ to trypsin, had little effect on TJ integrity and did not induce aberrant strands.

Depleting cell cholesterol alters calcium-induced assembly of tight junctions by monolayers of MDCK cells.

A role for lipids in the formation of tight junctions (TJ) has been proposed. Attempts to relate changes in whole cell phospholipid composition to the formation of TJs, however, have yielded equivocal results. The object in the present study was to relate changes in TJ of MDCK cells more specifically to alterations in plasma membrane lipids. Cholesterol, which resides primarily in the plasma membrane, was reduced by 25% after incubation of cell monolayers for 24 h in a low Ca2+ medium supplemented with (1-2 microM) Lovastatin, an inhibitor of hydroxymethylglutarylcoenzyme A (HMG-CoA) reductase. This was associated with a halving of the time required for Ca2+ to induce TJ formation as monitored by transepithelial electrical resistance (TER). [3H]Mannitol flux, and morphometric measurements made on freeze fracture replicas confirm that the effects on TER reflect changes in the characteristics of the paracellular pathway. Peak and steady state values of TER were also elevated over control values. The changes in cholesterol content and the time course for TJ assembly were apparent at levels of Lovastatin which do not affect prenylation of proteins, and were prevented if 5 mM mevalonate was present along with Lovastatin. Paradoxically, despite a decrease of approximately 1/3 in the Ca concentration required to yield maximum rates of TJ assembly, 45Ca2+ uptake was actually reduced after cholesterol depletion. The data suggest that cholesterol may modulate the properties of membrane proteins and/or phospholipids which interact with Ca2+, possibly on the exoplasmic leaflet, during TJ assembly.

Structure, function, and regulation of cellular tight junctions.

The tight junction (TJ) is a dynamic structure that is controlled, in part, by the activity of the cytoskeleton. It has become abundantly clear that, in the presence of Ca2+, assembly of the TJ is the result of cellular interactions that trigger a complex cascade of biochemical events that ultimately lead to the formation of an organized network of TJ elements, the composition of which remains unknown. The TJ functions both as a barrier between two fluid compartments and, to a lesser extent, as a fence between apical and basolateral membrane domains. To meet the many physiological and pathological challenges to which epithelia and endothelia are subjected, the TJ must be capable of a rapid and coordinated response, which depends on complex regulatory mechanisms. The precise characterization of the mechanisms involved in the assembly and regulation of the TJ is an area of current active investigation. However, until the biochemical composition of this structure has been defined and its gene identified, the TJ will continue to be an elusive yet tantalizing challenge to the cell biologist.

Arterial infections due to Listeria monocytogenes: report of four cases and review of world literature.

Early diagnosis and successful antimicrobial therapy have diminished the frequency of embolomycotic aneurysms, but infected aortic and small vessel aneurysms, arteriosclerotic plaques, and prosthetic grafts are becoming more common. A broad spectrum of pathogens, including Staphylococcus, Salmonella, Enterobacteriaceae, Pseudomonas aeruginosa, and some unusual organisms, are associated with this change. We treated four patients (three with abdominal aortic aneurysms and one with a prosthetic graft) with arterial infections caused by Listeria monocytogenes. Only seven other cases have previously been recorded in the world literature. Infection is suspected when a palpable or radiographically defined aneurysm is present with an otherwise obscure febrile illness. In about one-third of patients, blood cultures have yielded the pathogen. Newer imaging techniques have helped confirm the diagnosis. These infections are best managed by surgical resection in combination with long-term, appropriate antimicrobial therapy with ampicillin or sulfonamides. Unlike other adult listerial infection, except endocarditis, in arterial infection, immunosuppression and malignancy are not predisposing factors.

Inhibition of adenine nucleotide synthesis: effect on tight junction structure and function of clone 4 MDCK cells.

The formation and maintenance of tight junctions as a barrier to the diffusion of ions and other water-soluble across epithelia is an energy-dependent process. The administration of N-formyl-hydroxyaminoacetic acid (Hadacidin), an analog of aspartate and a competitive inhibitor of adenylosuccinate synthetase, has been shown to inhibit the multiplication of clone 4 MDCK cells and concomitantly reduce the levels of ATP and cAMP (J. Cell. Physiol. 140, 186-194 (1989)). When added to mitotically quiescent confluent cultures of clone 4 MDCK cells, millimolar concentrations of Hadacidin inhibited the generation of transepithelial electrical resistance (TER). In such cultures passive Na+ permeability was similar to controls indicating that the effect of Hadacidin was not on the transcellular pathway. That these cells were viable was demonstrated by their ability to exclude Trypan Blue, and the fact that they remained competent to develop steady state TER upon removal of the inhibitor. Suppression of TER was completely reversed within 48 h of replacing the Hadacidin-supplemented medium with one containing aspartate. Adenosine, but not aspartate, when added simultaneously with the drug, obviated the latter's effect on TER. A mixture of dibutyryl cAMP (db-cAMP) and theophylline was only partially effective in overcoming the effects of Hadacidin on the development of TER and, in fact, markedly delayed its development in control cultures not treated with the drug. When monolayers with established steady state TER were exposed to Hadacidin, no change was noted during the first 24 h. By 48 h, however, TER had decreased to very low values.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of native and chemically modified albumin with pulmonary microvascular endothelium.

The ability of native and chemically modified bovine serum albumin (BSA) to maintain normal pulmonary microvascular permeability was tested in "bloodless," fluorocarbon emulsion exchange transfused rats. Wet-to-dry weight ratios (W/D) of whole lung and morphometric estimates of the amount of ferritin transported to basement membrane were used to assess changes in water flux and macromolecular transport, respectively. Native and modified BSA in capillary walls were localized by immunogold techniques. Arginine residues of BSA were blocked with cyclohexanedione (CHD-BSA), and lysine residues were modified either by succinylation (Succ-BSA) or reductive methylation. Succinylation and CHD modification of BSA caused alterations in antigenicity and trypsin sensitivity; succinylation reduced the isoelectric point (pI). Whereas administration of either CHD-BSA or Succ-BSA increased the W/D, transport of ferritin to basement membrane was greater in the presence of Succ-BSA than CHD-BSA. By contrast, infusion of reductively methylated BSA in which modified lysines altered neither antigenicity nor pI, resulted in a W/D and amount of ferritin in basement membranes comparable to that of BSA. Binding of CHD-BSA and Succ-BSA to endothelial glycocalyx appeared to be reduced relative to native BSA and reductively methlyated BSA. The lowered pI of Succ-BSA may have contributed to its reduced binding; reductively methylated BSA with an unaltered pI was present in the glycocalyx. These data are consistent with a role for positively charged arginine residues in the interaction of albumin with the glycocalyx. The W/D of animals perfused with BSA was higher than those reported for rats perfused with complete rat serum proteins. This is consistent with the notion that serum factors, in addition to albumin, are required to maintain normal microvascular permeability.

Reduction of adenine nucleotide content of clone 4 MDCK cells: effects on multiplication, protein synthesis, and morphology.

The antitumor agent hadacidin (N-formyl-hydroxyamino-acetic acid), at 4 mM, inhibited the multiplication of clone 4 Madin Darby canine kidney (MDCK) cells within 24 hr. Growth resumed rapidly upon replacement of hadacidin with aspartate, an observation consistent with the drug's action as a competitive inhibitor of adenylosuccinate synthetase, an enzyme in adenine nucleotide biosynthesis. Data indicate that the drug-treated cells were arrested in S phase of the cell cycle. Accompanying inhibition of multiplication was a 16-fold increase in the area occupied by the cells and a refractoriness to release by treatment with trypsin. None of these changes occurred when 0.5 mM adenosine was included in the incubation mixture containing the inhibitor. Hadacidin decreased the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) content of the cells as well as the rate at which 3H-leucine was incorporated into protein. In the presence of 1 mM dibutyryl cAMP and theophylline, the drug had no effect on cell division and protein synthesis. The data suggest that, in clone 4 MDCK cells, the effects of hadacidin are mediated by diminishing the level of cAMP.

Diagnosis and treatment of pulmonary disorders of patients with the acquired immunodeficiency syndrome (AIDS).

It is apparent that the lung is frequently involved by a number of opportunistic pathogens and neoplasms in AIDS patients. What is even more disconcerting is the fact that often several infections or infection and neoplasm can coexist [236]. At this time, although effective therapy is at hand for most of the disorders mentioned, the patient's underlying immunodeficiency prevents any long-term survival and also usually leads to relapse when treatment is discontinued. Perhaps the use of newer antiviral compounds such as azidothymidine [237] or immunomodulating agents will help to reconstitute the waning immunocompetence and allow more durable responses in these currently fatal complications.

Modulation of tight junction formation in clone 4 MDCK cells by fatty acid supplementation.

Clone 4 MDCK cells, which generate a transepithelial electrical resistance (TER) of greater than 2,000 omega.cm2, were used to examine the role of membrane lipids in the barrier function of tight junctions. Phospholipid acyl groups were modified by supplementing cells grown in serum-free medium with 18:1(n-9), 18:3(n-3), or 18:3(n-6) complexed to albumin. Although both of these polyunsaturated fatty acids depress the melting point of membrane phospholipids, only 18:3(n-6) contributes significantly to eicosanoid production. Saturation indices of the phospholipids of cells supplemented with albumin alone, 18:1(n-9), 18:3(n-3), or 18:3(n-6) were 0.77, 0.78, 1.81, and 1.65, respectively. After trypsinization or removal of Ca2+, cells supplemented with 18:3(n-6) required longer periods of time to reestablish TER than did nonsupplemented cells or those incubated with 18:3(n-3) or 18:1(n-9). In contrast to MDCK strains I and II, clone 4 MDCK cells required continuous protein synthesis not only to reseal preexisting junctions after the addition of Ca2+ to Ca2+-depleted monolayers, but also to maintain steady-state TER. The rate of decay of TER in the presence of 1 microgram/ml of cycloheximide was 1.5 times greater in cells supplemented with either of the two 18:3 isomers than it was in nonsupplemented controls or in cells supplemented with 18:1(n-9). No significant difference was observed in the steady-state TER or selectivity of the tight junctions after fatty acid supplementation. These results suggest that there is a change in the dynamics of junction formation, rather than an alteration in their intrinsic properties.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism and incorporation into glycerolipids of exogenous 18:3(n-3) and 18:3(n-6) by MDCK cells.

The extent to which exogenous 18:3(n-3) and 18:3(n-6) were desaturated and elongated and the degree to which they and their derivatives altered the unsaturation index of cell glycerolipids were compared using clone 4 MDCK cells grown in lipid- and serum-free medium. Despite differences in the degree of unsaturation of the individual polyunsaturated fatty acids produced from 18:3(n-3) or 18:3(n-6), the unsaturation index of phospholipids increased similarly from 0.7 in control cells grown in serum- and lipid-free medium to ca. 1.6 in those supplemented with fatty acid. The added fatty acids had little effect on cell growth. The conversion of 18:3(n-6) to 20:3(n-6) and 20:4(n-6) was more rapid than that of 18:3(n-3) to 20:4(n-3) and 20:5(n-3). No significant quantities of 20:3(n-3) or 18:4(n-3) were noted. When both 18:3 isomers were supplied simultaneously, marked differences in the amounts of some species of n-3 and n-6 polyunsaturated fatty acids were observed. The presence of 18:3(n-6) and/or its derivatives suppressed levels of 20:4(n-3) and 20:5(n-3), perhaps through inhibition of the delta 6 and delta 5 desaturases responsible for their synthesis from 18:3(n-3). Similarly 18:3(n-3), and/or its longer more unsaturated derivatives, diminished the formation of 20:4(n-6) from 18:3(n-6). No marked effect on the products derived from elongation alone were observed.

Mortality and lung histopathology after inhalation lung injury. The effect of corticosteroids.

An animal model was used to study the effects of early administration of intramuscular corticosteroids on mortality and lung histopathology induced by a component of smoke. Thirty-six rabbits (mean weight, 2.7 kg) were exposed to acrolein vapor for 15 min; 30 min later the animals were divided into 3 treatment groups. One group received saline placebo intramuscularly at 12-h intervals, a second group was treated intramuscularly with 100 mg methylprednisolone at 12-h intervals, and a third group was treated with a single 100-mg dose of methylprednisolone followed by doses of saline at 12-h intervals. The animals were studied for a 72-h period. There was a significantly lower mortality in the 2 steroid-treated groups than in the nontreated group. A scoring system was developed for evaluating observed histologic changes in the lung. No correlation was seen between survival and histologic score or between score and treatment. High scores for particular histologic features did not explain mortality nor did they predominate in untreated animals; "vascular congestion" was found to be greater in the steroid-treated group. The beneficial effects of steroids in reducing mortality after inhalation of a common smoke constituent was not associated with any evidence of attenuation of lung damage.