Biomedical subjects

H Helbig

Publications and source records attributed to H Helbig.

At least 55 records · Page 3Linked to original sources

PubMed1991

Gamma-interferon induces differential expression of HLA-DR, -DP and -DQ in human ciliary epithelial cells.

The antigen-specific activation of T-helper lymphocytes is dependent on the presentation of antigen in context with the gene products of the major histocompatibility complex class II (MHC II). Aberrant expression of MHC II on the ciliary epithelium has been observed in uveitic eyes which may enable these cells to specifically interact with lymphocytes and may play a role in ocular autoimmunity. Human MHC II consists of three subclasses termed HLA-DR, -DP and -DQ, which seem to be differentially regulated and may have different functions. The present study was initiated to investigate the dynamics of the differential MHC II expression on cultured human non-pigmented ciliary epithelial cells (NPE cells) in response to gamma-interferon (gamma-IFN) by means of immunohistochemistry. NPE cells grown in control tissue-culture medium did not express MHC class II. HLA-DR and -DP could be induced by incubation with 100 mu/ml gamma-IFN for 3 days. HLA-DQ was expressed only weakly and at higher doses of gamma-IFN (greater than or equal to 500 mu/ml) and longer incubation periods (greater than or equal to 5 days). After removal of the gamma-IFN stimulus, all three MHC II subclasses persisted for several days. The differential expression of HLA-DR and -DP as compared with HLA-DQ in response to gamma-IFN in the ciliary epithelium is similar to observations in other non-lymphoid ocular cells but appears to be different from the regulation of MHC II expression on lymphoid cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1991

Mammalian ciliary-body epithelial cells in culture produce transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) has recently been found in the aqueous humor. The present study was initiated to test whether the ciliary-body epithelium, the site of aqueous humor formation, is capable of producing TGF-beta. Human, rat and bovine ciliary epithelial cells were grown in tissue culture and their supernatants were tested for TGF-beta using a bioassay. After acid activation of the samples, TGF-beta activity was found in the supernatants of all three mammalian ciliary epithelial cells. Most of this activity could be blocked by a neutralizing antibody against TGF-beta type 2. Native supernatants did not contain detectable TGF-beta activity. Thus, the ciliary epithelium is capable of producing the inactive form of TGF-beta (mainly type 2) and may be a source of TGF-beta in the aqueous humor.

Animals↗

PubMed1991

[Active immune regulation by cells of the ciliary body].

Immunoregulation in the eye requires both an effective defense against exogenous pathogens and protection of the delicate anatomy of the eye against "innocent bystander destruction", which accompanies inflammatory reactions. We studied the role of uveal cells for local immunoregulation in the eye and investigated the effect of cultured Lewis rat ciliary body cells on antigen-specific 3[H]-thymidin incorporation in a T-helper-lymphocyte cell line (ThS) specific for the retinal-soluble antigen (SAg). Ciliary body cells inhibit the proliferation of ThS. This inhibition is not species-specific and is mediated by at least two mechanisms: a soluble inhibitor and a contact-requiring factor that is trypsin-sensitive, suggesting a protein molecule. After removal of these inhibitory components, ciliary body cells also exhibit the capacity to effectively present SAg to ThS. This dual effect of ciliary body cells on T-helper lymphocytes supports the concept of active local immunoregulation in the eye.

Animals↗

PubMed1991

[Differential expression of HLA DR, DP and DQ in cultivated, human ciliary body epithelial cells].

The antigen-specific activation of T-helper lymphocytes is dependent on the presentation of antigen in context with the gene products of major histocompatibility complex class II (MHC II). Aberrant expression of MHC II on the ciliary epithelium has been observed in uveitic eyes, which may enable these cells to interact specifically with lymphocytes and may play a role in ocular autoimmunity. Human MHC II consists of three subclasses, termed HLA DR, DP and DQ, which seem to be differentially regulated and may have different functions. The present study was initiated to investigate the dynamics of the differential MHC II expression on cultured human non-pigmented ciliary epithelial cells (NPE cells) in response to gamma-interferon by means of immunohistochemistry. NPE were isolated by a technique of selective adhesion to tissue culture substrate, and growth was induced after transfection with an origin-defective mutant of SV-40 virus DNA. NPE grown in control tissue culture medium did not express MHC class II. HLA DR and DP could be induced by incubation with low concentrations of gamma-IFN for short periods. HLA DQ was expressed only weakly even with higher doses of gamma-IFN and longer incubation periods. The differential expression of HLA DR and DP, compared to HLA DQ in response to gamma-IFN in the ciliary epithelium, is similar to observations in other non-lymphoid ocular cells, but appears to be different from the regulation of MHC II on lymphoid cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line, Transformed↗

PubMed1990

Dual effect of ciliary body cells on T lymphocyte proliferation.

The interaction between organ-resident cells from the anterior uvea of the eye and T helper (Th) cells was investigated. Cells from Lewis rat ciliary body processes (CB cells), grown in tissue culture using an explant technique, could be induced to express major histocompatibility complex class II (Ia) antigens by incubation with rat interferon-gamma. Ia+ CB cells only poorly functioned as antigen-presenting cells (APC) for a syngeneic, uveitogenic Th cell line specific for the retinal soluble antigen (SAg). Moreover, if added to an Ag-driven lymphocyte proliferation assay in the presence of conventional APC, the rat CB cells had an inhibiting effect on Th proliferation. This inhibitory activity was not species specific, since similar effects were observed with bovine and human ciliary epithelial cells. The suppressive activity of CB cells was composed of a soluble factor, as well as a membrane-associated inhibitor. The soluble activity did not appear to be related to transforming growth factor-beta (TGF-beta), since no reversal of inhibition by a neutralizing antibody to TGF-beta was found. Part of the soluble inhibitory activity could be reversed by indomethacin treatment. The membrane-associated component was trypsin sensitive, suggesting a protein molecule. After abrogation of the inhibitory capacity by trypsin treatment and fixation by glutaraldehyde, CB cells effectively presented SAg to Th cells. These data suggest that CB cells are capable of mediating both Ag presentation and inhibition of Th cell proliferation.

Animals↗

PubMed1990

Induction of MHC class II antigen in cultured bovine ciliary epithelial cells.

The expression of major histocompatibility complex (MHC) class II antigens in cultured bovine ciliary epithelial cells was investigated by means of indirect immunohistochemistry and immunocytofluorometry. Ciliary epithelial cells grown in control tissue-culture medium did not express MHC class II. However, after incubation with bovine gamma-interferon (IFN-G) in concentrations as low as 0.3 units/ml, nearly all cells stained for MHC class II. Tumor necrosis factor increased IFN-G-induced MHC class II expression. A reduction in IFN-G-induced MHC class II expression was observed with dexamethasone, prostaglandin E2 and alpha-interferon. To test whether MHC class II expression in response to IFN-G was specific for the ciliary epithelium, several intraocular tissues were grown in culture and incubated with IFN-G. MHC class II expression was observed in all tissues tested for response to IFN-G, but at different sensitivities. Retinal pigment epithelium and ciliary epithelium exhibited the highest sensitivity, followed by corneal endothelium and lens epithelium; the lowest sensitivity was observed for retinal vascular pericytes. The results are discussed in the context of MHC class II expression on the ciliary epithelium in anterior uveitis.

Animals↗

PubMed1990

Endotoxin-induced production of inflammatory mediators by cultured ciliary epithelial cells.

Systemic injection of bacterial endotoxin (Lipopolysaccharide, LPS) in experimental animals induces anterior uveitis without major pathological changes in other organs. The present study investigates the effect of LPS on production of inflammatory mediators in cultured bovine pigmented ciliary epithelial cells (CB-cells) by means of radioimmunoassays and bioassays. LPS was found to stimulate CB-cells to secrete prostaglandin E2 and prostacyclin (assayed as its stable metabolite 6-keto-prostaglandin F1a), but not leukotriene B4 or thromboxane A2 (assayed as its stable metabolite thromboxane B2). CB-cells produced membrane-associated interleukin 1-activity in response to LPS, but no tumor necrosis factor-activity was found after challenge of CB-cells with LPS. The direct effect of LPS on production of inflammatory mediators by cells from the anterior uvea could play a role in the pathophysiology of endotoxin-induced uveitis.

Animals↗

PubMed1990

Membrane voltage recordings in a cell line derived from human ciliary muscle.

A smooth muscle cell line (H7CM) was established from the ciliary muscle of a 1-day-old human infant. The cultured cells had a normal female karyotype (46 XX) and could be maintained in cell culture for at least 11 generations. A common feature of confluent cultures was the presence of abundant bundles of 6-7 nm microfilaments associated with dense bodies. Both the ultrastructural appearance and the presence of smooth muscle-specific alpha-isoactin (also present in the human ciliary muscle in situ) support the smooth muscle origin of the H7CM cell line. Continuous membrane voltage (Vm) recordings were obtained in confluent monolayers of H7CM cells using glass microelectrodes. Resting Vm in 105 impalements averaged -66.2 +/- 0.7 mV (mean +/- standard error of the mean). In this system, rapid membrane transients induced by changing of the superfusing test solutions were detectable. Relative K+ conductance was characterized, and the contribution of electrogenic sodium/potassium adenosine triphosphatase to Vm was investigated. Under control conditions, H7CM cells were electrically quiescent. However, action potentials could be induced by application of 10 mM barium. Barium-induced action potentials were not abolished by removal of extracellular Na+ nor were they inhibited by the presence of tetrodotoxin. However, they were blocked by verapamil, fulfilling criteria believed to be typical for smooth muscle cells. Acetylcholine, carbachol, and to a lesser extent pilocarpine induced a reversible Vm depolarization. The effect of acetylcholine was blocked by atropine, implying muscarinic receptor involvement in the Vm response. Collectively, these findings show the potential usefulness of cultured ciliary muscle cells in understanding further the cellular mechanisms underlying drug-induced contraction of the human ciliary muscle.

Acetylcholine↗

PubMed1990

[Mechanism of ascorbic acid transport in the aqueous humor].

The concentration of ascorbic acid in the aqueous humor in many species (including humans and oxen) is several times higher than the plasma concentration. The role of ascorbic acid as a protective mechanism in the intraocular tissues against light-induced damage is discussed. We have performed uptake studies using 14C-labeled ascorbic acid in cultured bovine pigmented ciliary epithelial cells in order to study the mechanisms for ascorbic acid transport from the ciliary body to the aqueous humor. We found that intracellular accumulation of ascorbic acid was Na(+)-dependent. Data from kinetic experiments suggest the presence of a co-transport system for 2 Na+ and 1 ascorbate. The oxidized form of the vitamin (dehydro-ascorbic acid, DHA) was taken up in the cell by a Na(+)-independent mechanism. Using thin-layer chromatographic assay, we found intracellularly only ascorbic acid after incubation with extracellular DHA, indicating the presence of DHA reductase activity in ciliary body epithelial cells. Thus, these cells possess two mechanisms for intracellular accumulation of ascorbic acid: (1) co-transport for 2 Na+ and 1 ascorbate and (2) a Na(+)-independent DHA transport in cooperation with intracellular DHA reductase. These mechanisms could be involved in the transport of ascorbic acid from the stroma to the aqueous humor.

Animals↗

PubMed1989

Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells.

The ciliary muscle which is involved in accommodation and regulation of aqueous humour outflow resistance resembles smooth muscle in other parts of the body. In the present investigation we used an established primary cell line (H7CM) to study the effects of endothelin, a novel vasoconstrictor peptide, on membrane voltage (V) and intracellular calcium in cultured human ciliary muscle cells. Membrane voltage was measured in confluent monolayers of H7CM cells using conventional microelectrodes. Intracellular calcium concentration [( Ca]i) was measured in single H7CM cells using the fluorescent calcium indicator fura-2. Under resting conditions V averaged -66.9 +/- 0.7 mV (mean +/- SEM, n = 125). Endothelin (10(-10)-10(-6)M) induced a dose-dependent reversible membrane voltage depolarization and a dose-dependent rise in [Ca]i. The initial calcium peak was followed by a recovery phase during which oscillations of [Ca]i occurred. The initial calcium peak was not dependent on the presence of extracellular calcium and was not abolished in the presence of the calcium antagonist verapamil (10(-4)M). Thus it is probably mediated by a release of calcium from intracellular reservoirs. We conclude that cultured human ciliary muscle cells express a functional endothelin receptor.

Benzofurans↗

PubMed1989

Electrogenic Na+-ascorbate cotransport in cultured bovine pigmented ciliary epithelial cells.

The high level of ascorbic acid (AA) in the aqueous humor of many mammals suggests an active transport of AA across the double-layered ciliary epithelium from blood to aqueous humor. We used [14C]AA to study AA uptake in bovine pigmented ciliary epithelial cells in tissue culture. We observed a 40-fold intracellular accumulation of AA, which was dependent on extracellular Na+. With labeled dehydroascorbate (DHA, the oxidized form of the vitamin) in the medium, there was a 20-fold intracellular accumulation of the label. However, the time course of DHA uptake was different compared with AA uptake and was not Na+ dependent, suggesting different transport systems for AA and DHA. AA uptake was inhibited by 1 mM phloretin and in the presence of isoascorbate. Furthermore, AA uptake was markedly reduced when intracellular Na+ was elevated by preincubation with ouabain or amphotericin B. With increasing AA concentration, Na+-dependent AA uptake exhibited first-order saturation kinetics with half-maximal uptake at 76 microM AA. Na+ dependence of AA uptake revealed a sigmoidal curve of Na+-dependent AA uptake vs. Na+ concentration with a half-maximal AA uptake at 45.4 mM Na+. The slope of the Hill plot from these data was 1.94, suggesting a transport system translocating two or more Na+ for one AA. This stoichiometry implies electrogenicity of the transporter. We, therefore, measured membrane potentials using conventional microelectrodes. Addition of 200 microM AA resulted in a depolarization of the membrane voltage by 4.9 +/- 0.5 mV (n = 22), which was absent in Na+ free medium and was markedly reduced by phloretin.(ABSTRACT TRUNCATED AT 250 WORDS)

Amphotericin B↗

PubMed1989

Continuous membrane voltage recordings in A10 vascular smooth muscle cells: effect of AVP.

Continuous membrane voltage (V) recordings were obtained in A10 vascular smooth muscle cells (rat aorta) using glass microelectrodes. Resting membrane voltage in 262 impalements averaged 54.0 +/- 0.4 (SE) mV. Relative K+ conductance was characterized, and the contribution of electrogenic Na+-K+-ATPase to membrane voltage was investigated. Action potentials could be induced by application of 1 mM barium or 10(-4) M acetylcholine. In a few recordings, spontaneous spike activity occurred, and this could be abolished by 5 mM MgCl2 or by removal of extracellular Ca2+. Barium-induced action potentials were not dependent on the presence of extracellular Na+ and not inhibitable by 10(-6) M tetrodotoxin. Application of 10(-6) M [Arg8] vasopressin (AVP) for 30 s caused a typical biphasic membrane voltage response with an initial transient hyperpolarization of -9.5 +/- 1.1 mV and a more sustained subsequent depolarizing response averaging 28.2 +/- 1.3 mV (mean +/- SE, n = 58). The effect of AVP on membrane voltage was blocked by the V1-antagonist [beta-mercapto-beta,beta-cyclopentamethylenepropionyl1,O-Me- Tyr2,Arg8]vasopressin. The initial hyperpolarizing component of the membrane voltage response to AVP became more prominent when V was predepolarized, for example, by a preceding AVP application. However, when AVP was applied during high K+ depolarization or in the presence of quinidine (1 mM), the initial hyperpolarizing response was practically abolished. The time course of the initial hyperpolarization was shown to be similar to the calcium transient observed in fura-2-loaded A10 cell suspensions after the application of AVP. We conclude that the initial AVP-induced hyperpolarization in A10 cells corresponds to an activation of Ca2+-activated K+ channels.

Acetylcholine↗

PubMed1989

Role of HCO3- in regulation of cytoplasmic pH in ciliary epithelial cells.

Cytoplasmic pH (pHi) was monitored using the pH-sensitive absorbance of 5(6)carboxy-4',5'-dimethylfluorescein in monolayers of a cell clone derived from bovine pigmented ciliary epithelium (PE) transformed with the simian virus 40. 1) Changing extracellular media from a nominally HCO3(-)-free solution to a solution containing 28 mM HCO3(-)-5% CO2 at constant extracellular pH (7.4) resulted in a delayed alkalinization of pHi, which was 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) sensitive and was inhibited in Na+-free medium and in Cl(-)-depleted cells. 2) DIDS pretreatment acidified pHi in HCO3(-)-containing media. 3) Replacing extracellular Cl- resulted in a DIDS-sensitive, HCO3(-)-dependent, and Na+-independent alkalinization. 4) Replacing extracellular Na+ in HCO3(-)-containing media led to a partly DIDS-sensitive intracellular acidification. 5) Recovery of pHi after an alkali load (acetate prepulse) had a HCO3(-)-dependent and DIDS-sensitive component. 6) Two Na+-dependent components participated in pHi regulation after an acid load (NH4+ prepulse) in HCO3(-)-containing solution. One was amiloride sensitive, the other was DIDS sensitive and was inhibited in HCO3(-)-free media and after Cl- depletion. We conclude that in cultured PE, in addition to Na+-H+ exchange, two HCO3-transporters participate in pHi regulation. Cl(-)-dependent Na+-HCO3-symport regulates pHi during steady state and after an acid load, and Na+-independent Cl(-)-HCO3-exchange is involved in pHi recovery after an alkali load.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1989

Sodium bicarbonate cotransport in cultured pigmented ciliary epithelial cells.

Uptake of 22Na+ was studied in cultured bovine pigmented ciliary epithelial cells (PE) in HCO3-containing media. Two components of Na+-uptake were stimulated by intracellular acidification (NH4+-prepulse): One was amiloride-sensitive, the other DIDS-sensitive. The amiloride-sensitive component of Na+-uptake probably represents Na+/H+-exchange, which has previously been characterized in PE. The second, DIDS-sensitive component stimulated by intracellular acidification, was Cl- and HCO3--dependent. We conclude that a stilbene-sensitive, Cl--dependent Na+-HCO3--cotransport is present in PE. This transport could play an important role in aqueous humor formation.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1989

Coupling of 22Na and 36Cl uptake in cultured pigmented ciliary epithelial cells: a proposed role for the isoenzymes of carbonic anhydrase.

Uptake studies with 22Na and 36Cl were performed in cultured bovine pigmented ciliary epithelial cells (PE) to investigate interdependence of Na+ and Cl- transport. (1) 22Na uptake into NaCl depleted cells was stimulated by Cl-. This stimulation was abolished by the simultaneous application of amiloride (1 mM) and bumetanide (0.1 mM), indicating two independent mechanism for Cl- stimulated Na+ uptake: loop diuretic sensitive Na+/Cl- symport and an indirect stimulation of Na+/H+ exchange by Cl-. The latter component of Cl- stimulated Na+ uptake was HCO3- dependent. (2) 36Cl uptake was increased by extracellular Na+. Na+-stimulated Cl- uptake also consisted of two components. One was bumetanide sensitive and the other was blockable by amiloride and partly inhibited by the carbonic anhydrase (CA) inhibitor methazolamide (0.1 mM). (3) Homogenized PE cells were tested for biochemical CA activity using an electrometric method. The cytoplasmic as well as the membrane fraction contained specific CA activity. (4) A model is presented for Na+ and Cl- transport into PE: in addition to Na+/Cl- symport, Na+/H+ and Cl-/HCO3- double exchange may operate in the ciliary epithelium. The latter mechanism provides NaCl uptake into the cell in exchange for H+ and HCO3-, which recycle as CO2 across the membrane. This recycling of CO2 and HCO3-/H+ (and hence indirectly NaCl uptake) is facilitated by the cooperation between membrane bound and cytoplasmic CA.

Amiloride↗

PubMed1989

Intracellular voltage recordings in bovine non-pigmented ciliary epithelial cells in primary culture.

Bovine non-pigmented ciliary epithelial cells (NPE) have been isolated by a technique of selective adhesion to tissue culture plastic. NPE cells in primary culture proliferated and maintained epithelial-like morphology for about 4 weeks in tissue culture medium containing 10% fetal calf serum. If grown for longer than 4 weeks in serum-containing medium, cells changed their morphology and became elongated and spindle-shaped. Membrane potentials were measured using conventional microelectrodes. In NPE cells of epithelial-like shape, replacing extracellular Na+ induced a transient hyperpolarization of the membrane potential, while in elongated cells of spindle-shaped morphology an immediate depolarization was observed. We therefore only used epithelial-like NPE for further experiments. In these cells the mean membrane potential was -40.3 +/- 0.5 mV (n = 36). Relative K+ conductance was increased by extracellular alkalinization. Removing extracellular K+ led to a depolarization and readdition of K+ to K+ depleted cells resulted in a hyperpolarization. Both voltage responses were sensitive to ouabain, indicating that Na+/K+ ATPase is inhibited by K+ replacement, and that there is overshoot-activation of the pump when K+ is readded. Extracellular Cl- replacement led to a DIDS sensitive, transient depolarization, which is compatible with a stilbene-sensitive Cl(-)-conductance. Removing HCO3- led to a Na+ dependent and DIDS-sensitive depolarization. However, the electrical response on replacement of extracellular Na+ was not influenced by DIDS or the extracellular HCO3(-)-concentration.

Acetylcholine↗

PubMed1989

Electrical membrane properties of a cell clone derived from human nonpigmented ciliary epithelium.

Intracellular potentials were measured in a SV-40 virus-transformed cell clone derived from human nonpigmented ciliary epithelium using the microelectrode technique. (1) Membrane potential averaged -50.2 mV (+/- 0.6, n = 207). (2) Increasing the extracellular K+ concentration depolarized the membrane voltage. The amplitude of this potential response was reduced in the presence of 1 mM Ba2+. (3) Superfusing the cells with a Ca2+-free solution containing 1 mM EGTA depolarized the intracellular potential and diminished the voltage response upon increasing extracellular K+. (4) Extracellular alkalinization hyperpolarized the membrane potential and increased the voltage amplitude on increasing extracellular K+. (5) Addition of ouabain immediately reduced the intracellular potential. Removing extracellular K+ depolarized membrane voltage, readdition of K+ after K+ depletion transiently hyperpolarized intracellular voltage. Both potential responses were inhibited in the presence of ouabain. (6) Replacing extracellular Cl- by cyclamate resulted in a transient depolarization followed by a hyperpolarization. In the presence of SITS or DIDS (greater than or equal to 0.1 mM) the electrical responses of the cell membrane to Cl- replacement were blocked. We conclude that cultured human nonpigmented ciliary epithelial cells possess an electrogenic Na+/K+-ATPase, a K+ conductance modulated by Ca2+ and pH, and a Cl- conductance sensitive to stilbene derivatives.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1989

[Carbonic anhydrase and its function in ion transport in cultivated pigmented ciliary body epithelial cells].

The effect of inhibitors of the carbonic anhydrase on aqueous humor formation suggests that carbonic anhydrase has an important role in ion transport across the ciliary epithelium. We therefore investigated the role of carbonic anhydrase in Na+ and Cl- transport in cultured bovine pigmented ciliary epithelial cells (PE) using the radioactive isotopes 22Na and 36Cl. Our findings can be summarized as follows. (1) Na+ uptake into the cell occurs via an amiloride-sensitive Na+/H+ exchanger. (2) Cl- is transported into the cell in exchange for bicarbonate ions. (3) PE cells contain biochemical carbonic anhydrase activity. (4) Na+ uptake into NaCl-depleted cells is markedly stimulated by Cl-. This stimulation is HCO3- dependent and is completely blocked by amiloride and partly inhibited by the carbonic anhydrase inhibitor methazolamide. (5) A model is introduced for transport of NaCl in the ciliary epithelium: carbonic anhydrase is responsible for coupling of Na+/H+ exchange and Cl-/HCO3- countertransport. This model could explain the effect of carbonic anhydrase inhibitors on intraocular pressure.

Animals↗