Henle Cells Research Articles

Biologic roles of the innermost cell layer of the outer root sheath in human anagen hair follicle: further electron microscopic study.

To elucidate the biologic roles and further cytologic characteristics of the innermost cell (IMC) layer of the outer root sheath (ORS), human anagen hair follicles were ultrastructurally examined. In the lower follicle, the transeversely running tonofilaments in the inner side of the cytoplasm of the IMCs showed a massive accumulation, facing the keratinized part of a Huxley's cell protruding through a Henle's pore. In a rare instance, a spindle-shaped cell was seen between the IMC layer and the keratinized Henle's layer. At the lower isthmus portion, some of the IMCs containing a large number of tonofilaments showed a partial degeneration of the inner side of the cytoplasm. More distally, intercellular spaces between the keratinized IMCs and keratinized Henle's cells were partly dilated and contained amorphous substances. It is suggested that the IMCs in the lower follicle may play a role to support and cover the inner hair structures, tightly as hoops of a barrel. In the isthmus portion, the IMCs may loosely support and guide the keratinized Henle's cells undergoing degeneration.

Na,K-ATPase Activity in Renal Tubule Cells From Milan Hypertensive Rats

Several abnormalities of cation transport have been described in the Milan hypertensive rats (MHS). In this study we examined Na,K-ATPase activity in proximal convoluted tubules (PCT) cells and medullary thick ascending limb of Henle cells (TAL) from MHS and from the Milan normotensive rats (MNS). Na,K-ATPase activity was determined as 32P-ATP hydrolysis in single tubule segments. Na,K-ATPase activity (pmol Pi/mm t/h) was significantly higher in MHS than MNS both in PCT (903 +/- 227 n = 8 v 506 +/- 285 n = 12) and TAL (4324 +/- 800 n = 5 v 3063 +/- 625 n = 5). Na,K-ATPase dependent respiration was determined in PCT cell from MNS and MHS. Under basal condition Na,K-ATPase dependent respiration (mumol O2/mg protein/h) was higher in MHS than in MNS (24.2 +/- 1.8 n = 5 v 16.1 +/- 0.4 n = 5). When the cells were Na loaded by amphotericin Na,K-ATPase dependent respiration increased significantly more in MHS than MNS (38.4 +/- 1.6 v 26.8 +/- 2.2 n = 4). Thus, Na,K-ATPase activity is higher in renal tubule cells both at normal intracellular Na and after the cells have been Na loaded. The results indicate that regulation of Na homeostasis in renal tubule cell is different in MHS and MNS.

Rabbit urinary tamm-horsfall glycoprotein. Chemical composition and tentative carbohydrate structure.

The urinary Tamm-Horsfall protein (THP) is the major glycoprotein secreted by the mammalian kidney. We recently isolated and immortalized thick ascending limb of Henle cells from rabbit kidney, which produce Tamm-Horsfall protein in cell culture in vitro. In order to further study the yet undefined functional role and biosynthetic pathways of this protein, we first re-examined the chemical composition and the carbohydrate structure of rabbit urinary Tamm-Horsfall protein. Using precipitation with 0.58 mol/l NaCl a protein was isolated from rabbit urine which showed extensive microheterogeneity and had an average molecular mass of 95 kDa. Deglycosylation of the protein led to a loss of microheterogeneity and yielded a molecular mass of 58-60 kDa. Amino-acid analysis of the native and deglycosylated protein revealed a lower cysteine (20 mol/mol THP) and a higher histidine (20 mol/mol THP) content than described previously. Chemical analysis of the carbohydrates showed a high glucosamine (50 mol/mol THP), galactose (43 mol/mol THP), and mannose (24 mol/mol THP) content. The amount of sialic acid was 15 mol/mol THP. Using lectins to identify the structure of the carbohydrate chains it was shown that rabbit Tamm-Horsfall protein possesses complex-type oligosaccharide chains with terminal sialic acid, beta-galactose, and probably alpha-fucose and chains of the mucin type. These results indicate that some of the cysteine residues in the polypeptide chain of THP can be replaced by histidine, suggesting a role of some cysteins in metal binding rather than intramolecular stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)

Extratubular Tamm-Horsfall protein deposits induced by ureteral obstruction in mice

The effects of unilateral ureteral obstruction were studied in mice. Obstruction for 24 hr led to the formation of extratubular Tamm-Horsfall protein (TH) aggregates within the renal interstitium and at the base of distal convoluted tubular (DCT) cells. These DCT deposits were shown by ultrastructural analysis to be entirely extracellular. They had the fibrillar substructure characteristic of TH and had not been seen after urinary obstruction in other species. As a consequence of retrograde flow of urine to glomeruli, obstruction also caused TH aggregates to form within Bowman's spaces. These glomerular casts of TH were detected throughout the 3-week period of study after the release of unilateral obstruction. High serum titers of IgG antibodies to TH developed in mice intradermally immunized with TH but were not observed after obstruction alone. Circulating anti-TH antibodies combined with TH present on the basal surfaces of the thick ascending limb of the loop of Henle cells and DCT cells to form immune complexes in situ. Interstitial inflammation in the areas surrounding subepithelial tubular immune deposits was not present in the kidneys of immunized mice and was not selectively induced by temporary obstruction. However, foci of inflammation were seen in all obstructed kidneys. At later times, inflammatory foci in previously obstructed kidneys were associated with progressive scarring, primarily in polar regions. The location and severity of these changes within kidneys produced by obstruction in immunized mice did not differ from those in unimmunized mice. The titers of anti-TH antibodies in immunized mice were not enhanced or depressed as a consequence of unilateral ureteral obstruction. These studies demonstrate that complete obstruction of urinary flow in the mouse for periods as short as 24 hr may lead to progressive segmental renal scarring. These studies further indicate that increasing the quantities of extracellular TH by obstruction does not facilitate inflammatory responses to TH immune complexes formed in situ. While exposure of renal tissue to highly toxic components of extravasated urine may play a crucial role in inflammatory responses, autoimmunity to TH was not implicated as a contributing factor by the present studies in mice.

Dibutyryl cyclic AMP inhibits transport dependent QO2 in cells isolated from the rabbit medullary ascending limb.

Because the medullary thick ascending limb of the loop of Henle is the target of several polypeptide hormones that stimulate adenyl cyclase in this nephron segment, we examined the effects of cyclic AMP on thick ascending limb of the loop of Henle cells isolated by enzymatic digestion and density gradient centrifugation from the outer medulla of the rabbit kidney. The functional parameter that was measured was transport dependent oxygen consumption. Oxygen consumption was measured using a polarographic oxygen electrode in a constant temperature chamber. We found that dibutyryl cyclic AMP inhibited oxygen consumption in a dose dependent way. Maximal inhibition was observed at a concentration of 10(-5) M. The effect of dibutyryl cyclic AMP was not present in the absence of either sodium, chloride or both implying that its effect is restricted to the sodium and chloride dependent oxygen consumption. The effect of dibutyryl cyclic AMP was additive to that of furosemide 10(-4) M while that of furosemide was not additive to that of cyclic AMP suggesting that the site of action of cyclic AMP is distal to that of furosemide. The effect of dibutyryl cyclic AMP was not additive to that of ouabain and was absent in cells where oxygen consumption was stimulated with amphotericin B in the absence of chloride indicating that it has no effect on Na-K-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of epithelial cell invasion in vitro byYersinia enterocoliticawith anticellular antibody

An antiserum prepared in rabbits against Henle 407 intestinal epithelial cells inhibited invasion of Henle and HeLa cells by three serotypes of Yersinia enterocolitica but was inactive with Vero cells which are of monkey rather than human origin. Purified immunoglobulins and IgG antibody were also inhibitory but univalent Fab fragments were inactive. A single precipitin line of identity was observed with Henle and HeLa cell extracts in an Ouchterlony immunodiffusion assay. The results provide additional evidence that bacterial invasion is dependent upon phagocytic activity of the epithelial cells.

The innermost cell layer of the outer root sheath in anagen hair follicle: Light and electron microscopic study

To investigate cell differentiation in the outer root sheath (ORS) of the human anagen hair follicle, scalp skin specimens from individuals with normal hair were examined using light and electron microscopes. In the bulbar portion, the ORS was composed of two cell layers. The cells in the outer layer gradually increased in number upwards and finally underwent so-called trichilemmal keratinization, which proceeded toward the hair canal. On the other hand, the inner cells in the bulb formed a single cell layer along the outside of Henle's layer during cell differentiation; this unique layer was referred to as the innermost cell (IMC) layer of the ORS. With the use of hematoxylin and eosin stain, at the suprabulbar portion, where Henle's cells were keratinizing, an eosinophilic substance was deposited in the inner (Henle's) side of the IMC cytoplasm. The IMCs gradually became entirely eosinophilic and often produced keratohyaline granules. Ultrastructurally, the IMCs of the ORS showed an oblong shape forming a regularly arranged single-cell layer along the keratinizing Henle's layer and accumulated tonofilaments in the cytoplasm. They produced a few small electron-dense keratohyaline granules and were keratinized at the level at which Henle's layer still preserved its cell structure. From these findings, it is suggested that there are two types of keratinization of the ORS: trichilemmal keratinization and IMC keratinization.

Histochemical localization of hormone sensitive adenylate cyclase in defined nephron epithelia in culture.

Distal nephron epithelia of defined anatomical origin were microdissected from rabbit kidneys and individually explanted into an in vitro culture system. The 7 day monolayers grown from four different nephron epithelia were studied for the presence and amount of adenylate cyclase reaction product. In each case basal adenylate cyclase was compared with the enzyme reaction product after stimulation by arginine vasopressin, calcitonin, parathyroid hormone (PTH) and isoproterenol. In cortical collecting tubule cultures, the reaction was stimulated by vasopressin greater than isoproterenol greater than calcitonin. PTH had no effect. In cortical thick ascending loop of Henle cells, the stimulation was by calcitonin greater than vasopressin = PTH. Isoproterenol had no effect. In medullary ascending loop epithelia, stimulation was by vasopressin = calcitonin. Neither isoproterenol nor PTH had an effect. These observations indicate that adenylate cyclase is histochemically demonstrable in cultivated cells from rabbit distal nephron segments and that the enzyme activation by hormones is differential according to the epithelium of origin.

Temperature-dependent expression of virulence genes in Shigella species.

The pathogenicity of Shigella spp. involves the ability of the bacteria to penetrate and replicate within the epithelial cells of the large intestine. Model systems for examining the virulence of shigellae employ Henle intestinal epithelial cells in tissue culture and an in vivo assay for virulence in guinea pig eyes (Sereny test). Using these systems, we studied the genetic and physiological bases for the ability of shigellae to invade epithelial cells. We found that expression of virulence in Shigella spp. is dependent on the temperature at which the bacteria are grown. When grown at 37 degrees C, strains of Shigella flexneri 2a, Shigella sonnei, and Shigella dysenteriae 1 were fully virulent and invaded Henle cells. They also produced keratoconjunctivitis in guinea pigs. When grown at 30 degrees C, the bacteria neither penetrated Henle cells nor produced conjunctivitis in the Sereny test and were phenotypically avirulent. Strains grown at 33 degrees C were only partially invasive in the Henle assay, whereas strains grown at 35 degrees C were as invasive as strains grown at 37 degrees C. Using the Henle cell assay, we determined that the loss of ability to penetrate epithelial cells was completely reversed by shifting the growth temperature from 30 to 37 degrees C. The percentage of Henle cells invaded by bacteria increased with increasing time of growth at 37 degrees C. Restoration of invasiveness after growth at 30 degrees C required protein synthesis. When shigellae were grown at 30 degrees C and shifted to 37 degrees C for 2 h in the presence of chloramphenicol, the bacteria remained noninvasive. Similarly treated bacteria grown at 37 degrees C were still invasive. These results suggested that expression of one or more genes required for virulence of Shigella spp. are subject to regulation by growth temperature.

Attachment of Salmonella to mammalian cells in vitro.

The attachment of Salmonella typhimurium strain PHL67342 to several mammalian tissue culture cell lines was investigated. Strain PHL67342 failed to attach in significant numbers to the Buffalo green monkey (BGM), swine testicular (ST), and HeLa cell lines. Significant attachment was observed with the Henle intestinal cell line. Log-phase cells of strain PHL67342 attached in greatest numbers to the Henle cells after 45 min of incubation at 37 degrees C. Attachment to the Henle cells was not affected by D-mannose or D-galactose, but was markedly inhibited by high concentrations of alpha-methyl-D-mannoside. Also, Salmonella lipopolysaccharide had no effect on the attachment of strain PHL67342 to the Henle cells. Fimbriae were not detected on the bacterial cells used in the adherence experiments. These results suggest that some bacterial factor(s) other than fimbriae and lipopolysaccharide mediate the attachment of strain PHL67342 to the Henle cells.

Nephron epithelia in culture: Growth of loop of Henle cells in hormonally defined serum-free media

Nephron epithelia in culture: Growth of loop of Henle cells in hormonally defined serum-free media

Shigella Infection of Henle Intestinal Epithelial Cells: Role of the Host Cell

The process of Henle 407 embryonic intestinal epithelial cell infection by Shigella flexneri 2a M42-43 was studied in an in vitro model system. The role of the Henle cell was assessed. It was established that entry of S. flexneri into cells was suppressed by reagents which inhibit uptake of particles by phagocytic cells. The compounds tested included cytochalasin B, dibutyryl-cyclic adenosine monophosphate, choleragen (Vibrio cholera enterotoxin), iodoacetate, and dinitrophenol. Cytochalasin B inhibited infection at concentrations of 1.0 mug/ml or greater. Dibutyryl-cyclic adenosine monophosphate at concentrations of 1 mM and choleragen at 0.1 mug/ml caused significant suppression of infection. Iodoacetate or dinitrophenol, at 0.1 mM concentrations, inhibited internalization of virulent shigellae, and a combination of these compounds inhibited infection at 0.01 mM concentrations. Preincubation of Henle cell monolayers with the combination of iodoacetate and dinitrophenol (0.05 mM) also inhibited infection markedly. The data suggest that infection of epithelial cells by S. flexneri in vitro is accomplished by an endocytic process induced by virulent bacteria. The process appears to be similar to uptake of particles by phagocytic cells. Ultrastructural analysis by transmission electron microscopy provided corroborative evidence of phagocytosis of shigellae by Henle cells in that intracellular bacteria were often observed within membrane-limiting vacuoles resembling phagosomes.

Plasma membrane differentiations of keratinizing cells of the wool follicle: I. Gap junctions

Tight junctions in the keratinizing regions of the wool follicle have been studied primarily by the freeze-etch method. The evidence obtained indicates that tight junction zonulae are present in the plasma membranes of apposed Henle's cells, Henle's/Huxley's cells, and Henle's/companion cells. These zonulae were found from a region level with the middle of the dermal papilla up to where the inner root sheath is finally entirely hardened. From this, it is hypothesized that a function of Henle's layer is to seal off the extracellular space of the outer root sheath from that of the differentiating inner root sheath—fiber complex. The presence of both desmosomes and gap junctions within the tight junction zonulae was noted.

Plasma membrane differentiations of keratinizing cells of the wool follicle: III. Tight junctions

Tight junctions in the keratinizing regions of the wool follicle have been studied primarily by the freeze-etch method. The evidence obtained indicates that tight junction zonulae are present in the plasma membranes of apposed Henle's cells, Henle's/Huxley's cells, and Henle's/companion cells. These zonulae were found from a region level with the middle of the dermal papilla up to where the inner root sheath is finally entirely hardened. From this, it is hypothesized that a function of Henle's layer is to seal off the extracellular space of the outer root sheath from that of the differentiating inner root sheath—fiber complex. The presence of both desmosomes and gap junctions within the tight junction zonulae was noted.

Plasma membrane differentiations of keratinizing cells of the wool follicle: II. Desmosomes

Desmosomes have been studied in selected cell lines in the keratinizing regions of the Romney wool follicle using thin section and freeze-etch methods. The numbers of desmosomes increased during differentiation of the cortex, Henle's layer, and the fiber cuticle. Henle's layer desmosomes were larger than cortical desmosomes at a level above the dermal papilla and were incorporated in part, at least, into the hardened cell surface. Cortical desmosomes reached their maximum numbers in the region above the dermal papilla, where they constituted about 5% of the plasma membrane surface area. Both cortical desmosomes and those associated with fiber cuticle cells dedifferentiated in the region where Henle's cells harden. A possible sequence of events during the formation and dedifferentiation of cortical desmosomes is presented. The role of desmosomes in intercellular adhesion and as a site for the attachment of cytoplasmic fibrils, particularly early macrofibrils, is discussed.

Effects of prednisolone on phospholipid metabolism in tissue culture

The effect of prednisolone on the incorporation of 32P-phosphoric acid into asynchronously and synchronously growing Henle cells was determined. A 24 h exposure of cell cultures to prednisolone was necessary to produce a significant inhibition of total 32P incorporation into the cells. However, less than 1 h exposure to the hormone was sufficient to show an inhibition of 32P incorporation into the lipid fraction. After the separation of the phospholipid components by thin-layer chromatography, it was found that prednisolone caused no significant alteration in the distribution of the 32P label among the phosphatides. The incorporation of 32P into the phospholipid fraction of synchronously growing cells was suppressed by prednisolone during the entire division cycle.