Neutrophil transepithelial migration: regulation at the apical epithelial surface by Fc-mediated events.

Neutrophil (PMN) transepithelial migration is a major effector of epithelial defense in inflammatory diseases involving mucosal surfaces. However, major receptor-ligand interactions between epithelial cells and PMN remain incompletely characterized. To better define the molecular events involved in PMN interactions with epithelial cells, we produced a monoclonal antibody called g82 that inhibited PMN transepithelial migration in the physiological basolateral-to-apical direction. The g82 antigen localized to the apical surface of human colonic epithelium and was significantly upregulated under inflammatory conditions. Immunoprecipitation revealed two polypeptides of M(r) 207 and 32 kDa. F(ab')(2) fragments from g82 IgG had no effect on transmigration, suggesting Fc dependence. Further experiments confirmed dependence on the PMN Fc receptor CD32A and that the observed effects were secondary to a failure of PMN to detach from the apical epithelial surface. These Fc-mediated events were epitope specific since binding, isotype-matched antibodies did not affect detachment. These results identify a new mechanism for retention of PMN at the apical epithelial surface following transepithelial migration. This pathway may be important in pathogen clearance and mucosal pathophysiology associated with autoimmunity.

Human junction adhesion molecule regulates tight junction resealing in epithelia.

Epithelial cells form a highly selective barrier and line many organs. The epithelial barrier is maintained by closely apposed cell-cell contacts containing tight junctions, the regulation of which is incompletely understood. Here we report the cloning, tissue localization and evidence for a role in epithelial barrier regulation of an immunoglobulin superfamily member that likely represents the human homolog of murine junction adhesion molecule (JAM). Analysis of the primary structure of human JAM, cloned from T84 epithelial cells, predicts a transmembrane protein with an extracellular domain that contains two IgV loops. Monoclonal antibodies generated against the putative extracellular domain were reactive with a 35-39 kDa protein from both T84 epithelial cells and human neutrophils. By immunofluorescence, JAM mAbs labeled epithelial cells from intestine, lung, and kidney, prominently in the region of tight junctions (co-localization with occludin) and also along lateral cell membranes below the tight junctions. Flow cytometric studies confirmed predominant JAM expression in epithelial cells but also revealed expression on endothelial and hematopoietic cells of all lineages. Functional studies demonstrated that JAM specific mAbs markedly inhibited transepithelial resistance recovery of T84 monolayers after disruption of intercellular junctions (including tight junctions) by transient calcium depletion. Morphologic analysis revealed that, after disassembly of cell-cell junctions, anti-JAM inhibition of barrier function recovery correlated with a loss of both occludin and JAM, but not ZO-1, in reassembling tight junction structure. Reassembly of the major adherens junction component E-cadherin was not affected by JAM specific mAbs. Our findings suggest that JAM plays an important role in the regulation of tight junction assembly in epithelia. Furthermore, these JAM-mediated effects may occur by either direct, or indirect interactions with occludin.

Cardiac integrins the ties that bind.

An elaborate series of morphogenetic events must be precisely coordinated during development to promote the formation of the elaborate three-dimensional structure of the normal heart. In this study we focus on discussing how interconnections between the cardiac myocyte and its surrounding environment regulate cardiac form and function. In vitro experiments from our laboratories provide direct evidence that cardiac cell shape is regulated by a dynamic interaction between constituents of the extracellular matrix (ECM) and by specific members of the integrin family of matrix receptors. Our data indicates that phenotypic information is stored in the tertiary structure and chemical identity of the ECM. This information appears to be actively communicated and transduced by the α1ß1 integrin molecule into an intracellular signal that regulates cardiac cell shape and myofibrillar organization. In this study we have assessed the phenotypic consequences of suppressing the expression and accumulation of the α1 integrin molecule in aligned cultures of cardiac myocytes. In related experiments we have examined how the overexpression of α2 and α5 integrin, integrins normally not present or present at very low copy number on the cell surface of neonatal cardiac myocytes, affect cardiac protein metabolism. We also consider how biochemical signals and the mechanical signals mediated by the integrins may converge on common intracellular signaling pathways in the heart. Experiments with the whole embryo culture system indicate that angiotensin II, a peptide that carries information concerning cardiac load, plays a role in controling cardiac looping and the proliferation of myofibrils during development.

Effect of tear lubricating solutions on in vivo corneal epithelial permeability.

PURPOSE: The rabbit corneal epithelial permeability was measured noninvasively following exposure to commercially available tear lubricating solutions. METHODS: The normal unanesthetized rabbit's corneal surface was either bathed in the tear lubricating solution for 5 min or received multiple applications of 2 drops per 30 min for 6 h for one or five days. The corneal epithelial permeability to carboxyfluorescein after a 5-min bath was measured with the Fluorotron Master. Fifteen commercially available tear preparations were tested. RESULTS: The baseline corneal epithelial permeability was 0.0455 +/- 0.0114 nm/s. The epithelial permeability values for the corneas bathed for 5 min in control solution (BSSplus) was 0.0798 +/- 0.0074 nm/s, in the preservative-free tear lubricating solutions the range was 0.512 to 0.542 nm/s, and in the preserved tear lubricating solutions the range was 0.3518 to 11.8873 nm/s. The preservative-free tear lubricating solutions had epithelial cell permeabilities up to 6 times greater than the control solution. Whereas, the preserved tear lubricating solutions had epithelial cell permeabilities up to 149 times greater than the control solution. Multiple applications of the tear lubricating solutions for 5 days were less damaging to the epithelial permeability than the 5 min bath applications. The resulting epithelial permeabilities were up to 2 fold greater than the control with the corresponding preservative-free solution and up to 29 fold greater than control with the corresponding preserved solution. CONCLUSION: An extended exposure of 5 min to various tear lubricating solutions demonstrated significant differences in epithelial cell permeability between preserved solutions and unpreserved solutions, whereas the multiple drop application technique demonstrated all of the preservative-free solutions and some of the preserved solutions to be insignificantly different from the control solution.

Role of the alpha 1 beta 1 integrin complex in collagen gel contraction in vitro by fibroblasts.

Matrix remodeling, critical to embryonic morphogenesis and wound healing, is dependent on the expression of matrix components, their receptors, and matrix proteases. The collagen gel assay has provided an effective model for the examination of the functional role(s) of each of these groups of molecules in matrix remodeling. Previous investigations have indicated that collagen gel contraction involves the beta 1 integrin family of matrix receptors and is stimulated by several growth factors, including TGF-beta, PDGF, and angiotensin II. In particular, collagen gel remodeling by human cells involves the alpha 2 beta 1 and, to a lesser extent, the alpha 1 beta 1 integrin complexes. The present studies were undertaken to determine the role of the alpha 1 integrin chain, a collagen/laminin receptor, in collagen gel contraction by rodent and avian fibroblasts. A high degree of correlation was found between the expression of the alpha 1 beta 1 integrin complex and the relative ability of cells to contract collagen gels. Further studies using antibodies and antisense oligonucleotides against the alpha 1 integrin indicated a significant role for this integrin chain in contraction of collagen gels by rat cardiac fibroblasts. In addition, antibodies to the alpha 1 integrin chain inhibited migration of these fibroblasts on a collagen substratum, suggesting that at least one role of this integrin is in migration of cells in collagen gels. These results indicate that the alpha 1 beta 1 integrin complex plays a significant role in cellular interactions with interstitial collagen that are involved in matrix remodeling such as is seen during morphogenesis and wound healing.

Safety and efficacy of diclofenac sodium 0.1% ophthalmic solution in acute seasonal allergic conjunctivitis.

A randomized clinical trial was conducted to compare diclofenac sodium 0.1% ophthalmic solution to placebo in relieving ocular signs and symptoms in patients with acute seasonal allergic conjunctivitis. Twenty patients (10 per treatment) qualified for this two week, double-masked study with moderate itching, bulbar conjunctival injection and a positive skin test. Diclofenac was statistically and clinically superior in the physician's global evaluation (p = 0.03) and the primary composite score [itching + bulbar/palpebral conjunctival injection (p = 0.037)] after two weeks of treatment. Four patients experienced some transient ocular burning/stinging with diclofenac. Diclofenac sodium appears to be effective for relieving the ocular signs and symptoms associated with acute seasonal allergic conjunctivitis.

A limited comparison of apraclonidine's dose response in subjects with normal or increased intraocular pressure.

We performed a multicentered, placebo-controlled, randomized, crossover study comparing the efficacy of 0.5% and 1.0% apraclonidine hydrochloride in 15 normal volunteers and 17 subjects with increased intraocular pressure. Apraclonidine 1% produced a maximum 30.4% +/- 14.0% (4.7 +/- 2.4 mm Hg) decrease in mean intraocular pressure in normal eyes and a 31.3% +/- 16.5% (7.6 +/- 4.2 mm Hg) decrease in eyes with increased pressure. Apraclonidine 0.5% produced a maximum 25.8% +/- 9.7% (4.0 +/- 1.7 mm Hg) decrease in mean intraocular pressure in normal eyes and a 27.4% +/- 16.0% (6.8 +/- 4.5 mm Hg) decrease in eyes with increased pressure. There was no statistically significant difference in mean percent intraocular pressure lowering effect between the 0.5% and 1.0% apraclonidine concentrations. Most subjects treated with apraclonidine had a greater than or equal to 20% reduction in intraocular pressure from baseline. Twelve hours after instillation of apraclonidine, nine of the normal volunteers had an intraocular pressure of 10 mm Hg or less. Apraclonidine produced the same percent intraocular pressure decrease regardless of the initial level of intraocular pressure.

Suprofen treatment of contact lens-associated giant papillary conjunctivitis.

This multicenter study of patients with contact lens-associated giant papillary conjunctivitis (GPC) was a randomized, double-masked comparison of a 1.0% suprofen solution versus the suprofen vehicle solution (placebo). Patients were given two drops of medication four times daily for up to 28 days and were clinically examined on days 0, 2, 7, 14, 21, and 28. The physicians' clinical judgments of the patients' responses to therapy significantly favored suprofen over placebo at day 21 (P = 0.02), while strongly favoring suprofen at day 14 (P = 0.057) and at day 28 (P = 0.067). The patients' opinions of their response to therapy significantly favored suprofen on day 14 (P = 0.03); a trend for suprofen was evident on day 28 (P = 0.1). Treatment with suprofen led to a greater overall reduction in ocular signs and symptoms than with placebo. Strong trends approaching statistically significant levels were found for reductions in the principal ocular sign, papillae, at day 28 (P = 0.068) and in mucus strands at days 14 and 28 (P = 0.09), which also favored suprofen.

Reduction of pupillary constriction during cataract surgery using suprofen.

The efficacy of a new nonsteroidal anti-inflammatory agent, suprofen, for reducing pupillary constriction during cataract surgery was ascertained in a double-masked, multicenter, clinical study. Prior to surgery 1.0% suprofen or a placebo was instilled; the surgeon's normal regimen of mydriatics and cycloplegics was used. Suprofen (209 patients) was far more effective than the placebo (203 patients) in maintaining a dilated pupil prior to intraocular lens (IOL) implantation (or instillation of a miotic). The mean pupillary area prior to IOL implantation was 6.3 sq mm larger (20% larger) in patients treated with suprofen than in patients receiving the placebo. The investigators' subjective evaluations of the adequacy of pupil size for IOL implantation and of the difficulty of IOL implantation favored patients treated with suprofen over those receiving the placebo.

Supraoptic neurons in the rat hypothalamo-neurohypophysial explant: double-labeling with Lucifer Yellow injection and immunocytochemical identification of vasopressin- and neurophysin-containing neuroendocrine cells.

By combining intracellular electrophysiology with double-labeled intracellular dye-marking and immunocytochemical identification of the same magnocellular neuroendocrine cell, we studied supraoptic neurons in the rat hypothalamo-neurohypophysial explant in vitro. This report examines neurophysiological and light microscopical features of vasopressin- and neurophysin-immunoreactive, pituitary-projecting supraoptic neurons.

Thermosensitive characteristics of a preoptic area neuron recorded over a 20 day period in the rabbit.

The thermosensitive characteristics of a single preoptic area neuron were monitored over a period of twenty days from a rabbit fitted with chronic recording electrodes. No demonstrable daily changes were detected in either the basal firing rate or the mean interspike interval during control recordings. Only minor daily variations were observed in thermosensitivity (impulses-second/degree C) and in the interspike interval coefficient of variation for this neuron in response to preoptic heating and cooling with a water-perfused thermode. When measured during early morning, early afternoon and at late night, thermosensitivity remained constant and showed no apparent circadian rhythm. The results from this single thermosensitive preoptic area neuron suggest that in spite of circadian rhythms of body temperature and other physiological parameters, some thermoregulatory neurons retain fixed temperature sensitive properties under conditions of stable ambient temperature.

Light- and electron-microscopic characterization of electrophysiologically-identified, horseradish peroxidase-injected magnocellular neuroendocrine cells in goldfish preoptic nucleus.

We recorded intracellularly from neurons in the goldfish preoptic nucleus which were antidromically identified by electrical stimulation of the pituitary gland and marked by intracellular injection of horseradish peroxidase for subsequent localization. At the light-microscopic level, labeled neurons resembled profiles of Golgi-impregnated neurons and lay in the magnocellular portion of the preoptic nucleus. Densely labeled axons and dendrites projected to the lateral forebrain bundle, the medial forebrain bundle, fiber tracts in the preoptico-hypophysial tract, small blood vessels and capillaries, the ependymal lining of the third ventricle and toward the preoptic neurons. Occasionally, a lightly-labeled, large perikaryon lay adjacent to a large, heavily-labeled magnocellular neuron. Ultrastructural examination of these identified cells revealed dense reaction product in neuronal perikarya and processes. Heavily labeled perikarya had elaborate networks of endoplasmic reticulum, extensive Golgi apparatus, occasional somatic spines and infrequent axo-somatic contacts from unlabeled neurons. These labeled perikarya which were frequently in close somatic apposition with unlabeled profiles were sometimes adjacent to a large, lightly-labeled perikaryon. A thin glial sheath separated most labeled neurons and processes from brain capillary endothelium. Labeled dendrites had heavily labeled spines and axo-dendritic contacts from unlabeled neurons. Labeled axons abutted unlabeled-axons and -dendrites. Synaptic boutons innervating labeled structures always contained small clear synaptic vesicles and some boutons also contained large dense-core vesicles. These results demonstrate the complex connections of goldfish preoptic magnocellular neuroendocrine cells with other neurons, fiber systems, brain capillaries, ventricular ependyma and the pituitary and provide further support for non-endocrine as well as endocrine functions of magnocellular neurons.

A technique combining intracellular dye-marking, immunocytochemical identification and ultrastructural analysis of physiologically identified single neurons.

A new method which produces an insoluble osmophilic polymer within Lucifer Yellow-injected neurons has allowed us to develop a technique for the ultrastructural examination of electrophysiologically characterized, immunocytochemically identified single neurons. In this initial report, we examine the light- and electron-microscopic features of neurophysin-containing, pituitary-projecting neurons in the goldfish nucleus.

The localization of vasotocin and neurophysin neurons in the diencephalon of the pigeon, Columba livia.

Vasotocin (VT)- and neurophysin (NP)-synthesizing neurons were demonstrated by immunocytochemistry in the diencephalon of the pigeon, Columba livia. Three diencephalic regions contain VT-NP cells: (1) periventricular preoptic area and hypothalamus, including nucleus periventricularis magnocellularis (PVM); (2) lateral preoptic area and hypothalamus; and (3) dorsal diencephalon. The immunoreactive cells in each of these three regions were divided into groups based on cytology and topography. No differences were found in the location of VT and NP cell groups. The periventricular region contains three continuous cell groups (P1-P3) extending from the posteroventral preoptic area to the anterodorsal hypothalamus and PVM. The lateral region has two cell groups composed of medium- to large-sized cells associated with the quintofrontal tract (L1) or with the optic tract (L2), while a third group (L3) lies between these two cell groups. Two accessory cell groups reside in the dorsolateral hypothalamus; L4 contains scattered cells of varied size, whereas L5 has small- to medium-sized cells clumped together. The dorsal diencephalic cell groups are found in the following locations: (1) lateral and dorsal to the lateral forebrain bundle (DD1); (2) in the area ventral to the dorsomedial anterior thalamic nucleus and dorsolateral to PVM (DD2); and (3) at the dorsolateral border of nucleus rotundus (DD3). To avoid potentially inaccurate mammalian homologies, the cell group nomenclature denotes topographic position. Nevertheless, the presence of VT-NP cells in PVM and projections to the brainstem and spinal cord suggest a homology between PVM and some of the parvocellular subnuclei of the mammalian paraventricular nucleus.

Ultrastructural immunocytochemical characterization of isotocin, vasotocin and neurophysin neurons in the magnocellular preoptic nucleus of the goldfish.

We describe the ultrastructural localization of isotocin, vasotocin and neurophysin in the magnocellular preoptic nucleus of the goldfish. With the aid of immunocytochemical techniques, we see staining both in classical neurosecretory granules and in diffuse agranular form throughout somata and processes. Signs of cellular and synaptic interactions between chemically identified neurons include axon terminals which contain vasotocin immunoreactivity and membrane specializations (puncta adhaerentia) between adjacent somata. Our investigations provide an anatomical basis for neuroendocrine and neurotransmitter-like functions of peptidergic neurons in the teleost preoptic nucleus.

Cholecystokinin in hippocampal pathways.

The distribution of cholecystokininlike (CCK-L) immunoreactive cells and fibers in the rat hippocampal formation and its afferent and efferent connections was studied using the immunoperoxidase technique. In the hippocampal formation CCK-L immunoreactive perikarya were located in the polymorphic zone of the dentate hilus, all layers of Ammon's horn, the subiculum, the presubiculum, and the entorhinal cortex. Cholecystokininlike immunoreactive fibers extended from cell bodies or were located around the cell bodies in the entorhinal cortex, subiculum and stratum pyramidale of Ammon's horn, and among the granule cells and inner molecular layer of the dentate gyrus. The immunoreactive cells in the stratum oriens may be a type of basket cell, since processes from these cells extend into stratum pyramidale and collections of CCK-L immunoreactive fibers are seen around cell bodies in stratum pyramidale. Cholecystokininlike immunoreactive fibers were also observed in the alveus, ventral and lateral fimbria, and ventrolateral lateral septal nucleus. Some of these immunoreactive fibers, therefore, being to either an efferent or afferent hippocampal pathway(s) originating from CCK-L immunoreactive pyramidal cells in the hippocampal formation and/or from the hippocampal subcortical nuclei, the supramammillary nucleus, and the dorsomedial hypothalamic nucleus which contain CCK-L immunoreactive perikarya. The distribution of these immunoreactive fibers in the fimbria and lateral septal nucleus is most consistent with an anteriorly directed efferent hippocampal pathway.

Ultrastructural immunocytochemical localization of enkephalin in the goldfish preoptic nucleus.

This study describes the ultrastructural localization of the opioid peptide enkephalin (ENK) in the preoptic nucleus of the goldfish. Using immunocytochemical techniques, ENK could be seen in neurosecretory granules and throughout the cytoplasm of magnocellular neurons with an agranular distribution. ENK was also associated with small clear vesicles and large dense-core vesicles within certain axon terminals in the preoptic nucleus. In this report, we discuss the cellular and synaptic relationships of ENK neurons in the preoptic nucleus of the teleost.

Dye-marked paraventricular neuroendocrine cells in vivo in cat hypothalamus.

In antidromically identified neurons in the cat hypothalamus we recorded and injected fluorescent dye-markers (Lucifer Yellow, LY; Procion Yellow, PY) intracellularly. The dye-filled neurons lay in the rostral portion of the magnocellular paraventricular nucleus of the hypothalamus. We observed two morphological cell types of similar size based on the intracellular injections of LY or PY: a bipolar cell type with fusiform perikaryon and a multipolar cell type with a polygonal perikaryon. These morphological cell types correspond to previous descriptions of immunocytochemically identified vasopressinergic and oxytocinergic magnocellular neurons in mammals. This study demonstrates the feasibility of in vivo intracellular dye-marking and electrophysiological recordings from mammalian hypothalamic neurons. We have here a basis for correlating morphological characteristics with the physiological traits of single magnocellular neuroendocrine cells.

Osmotic regulation of vasopressin in the cat.

This study examines the effects of blood osmolality on the release of arginine vasopressin (AVP) in the cat. Prior to the beginning of the experiments, the chamber-isolated, unanesthetized cat, allowed water ad libitum had a constant plasma osmolality averaging 320 +/- 2 (SE) mosmol/kg and a constant plasma AVP averaging 3.4 +/- 0.7 microU/ml. Water loading decreased plasma osmolality to 312 +/- 2 mosmol/kg and lowered plasma AVP to 1.3 +/- 0.2 microU/ml. As dehydration occurred during fluid restriction, the plasma osmolality increased and plasma AVP rose to 8 times the base line after 2 days. The rise in plasma AVP correlated linearly with the rise in plasma osmolality (r = 0.81; P less than 0.01). The cat's osmotic-vasopressin relationships are unique among mammals, revealing an elevated osmotic "set point" (threshold) and with regression analysis an increased "gain" or "'sensitivity" (increased slope of the regression line). We speculate that these unusual osmotic-AVP relationships may be related to some specialized features of the cat, such as hypothalamic anatomy or cerebral arterial blood supply.