Biomedical subjects

G M Roomans

Publications and source records attributed to G M Roomans.

At least 37 records · Page 2Linked to original sources

PubMed2001

Effect of luminal osmolarity on ion content of connective tissue in rat trachea after epithelial damage.

The authors investigated the physical role of the airway epithelium in response to changes in the airway surface fluid's composition by superperfusing the lumen with nonisotonic solutions. Morphological studies and measurements of changes in ion content in the underlying connective tissue (CT) were carried out. The study used an experimental model of isolated rat trachea. The trachea was mounted in an extraluminal organ bath with Ringer's solution, whereas the lumen was perfused with a fluid in which the NaCl concentration was varied. The tissue was fixed for electron microscopy or frozen for X-ray microanalysis. X-ray microanalysis showed that the Na and Cl content of the CT increased with increasing luminal NaCl concentrations. This increase was significantly larger when the tight junctions had been damaged by exposure to ethylene glycol tetra-acetic acid. At high luminal NaCl concentrations, electron microscopy showed that a significant influx of fluid into the CT had occurred in tracheae with damaged epithelia. Damage to the epithelium also resulted in ultrastructural changes in myofibroblasts, increased diameter of capillaries, and thickening of the basement membrane. The epithelium evidently plays a crucial role in the regulation of the ion content of the connective tissue in the airway wall, and epithelial damage may explain the greater sensitivity to provocation with hyperosmolar sodium chloride solutions or airway dehydration observed in patients with asthma or cystic fibrosis.

Animals↗

PubMed2001

Pharmacological treatment of the ion transport defect in cystic fibrosis.

Cystic fibrosis (CF) is a lethal monogenetic disease characterised by impaired water and ion transport over epithelia. The lung pathology is fatal and causes death in 95% of CF patients. The genetic basis of the disease is a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated chloride channel. The most common mutation, DeltaF508, results in a protein that cannot properly be folded in the endoplasmic reticulum, is destroyed and hence does not reach the apical cell membrane. This paper will discuss those pharmacological approaches that are directed at correcting the defect in ion transport. At present, no clinically effective drug is available, although research has defined areas in which progress might be made. These are the following: (1) the drug 4-phenylbutyrate (4PBA) increases the expression of DeltaF508-CFTR in the cell membrane, probably by breaking the association between DeltaF508-CFTR and a chaperone; (2) a number of xanthines, in particular 8-cyclopentyl-1, 3-dipropylxanthine (CPX), are effective in activating CFTR, presumably by direct binding and also possibly by correcting the trafficking defect; (3) the isoflavone genistein can activate both wild-type and mutant CFTR, probably through direct binding to the channel; (4) purinergic agonists (ATP and UTP) can stimulate chloride secretion via a Ca(2+)-dependent chloride channel and in this way compensate for the defect in CFTR, but stable analogues will be required before this type of treatment has clinical significance; (5) treatment with inhaled amiloride may correct the excessive absorption of Na(+) ions and water by airway epithelial cells that appears connected to the defect in CFTR; although clinical tests have not been very successful so far, amiloride analogues with a longer half-life may give better results. The role of CFTR in bicarbonate secretion has not yet been established with certainty, but correction of the defect in bicarbonate secretion may be important in clinical treatment of the disease. Currently, major efforts are directed at developing a pharmacological treatment of the ion transport defect in CF, but much basic research remains to be done, in particular, with regard to the mechanism by which defective CFTR is removed in the endoplasmic reticulum by the ubiquitin-proteasome pathway, which is a central pathway in protein production and of significance for several other diseases apart from CF.

Amiloride↗

PubMed2000

X-ray microanalysis of intestinal chloride transport in vitro and in vivo.

Chloride secretion from crypt cells in mouse duodenum or jejunum was investigated in in vivo and in vitro models by electron probe X-ray microanalysis. IBMX-induced chloride secretion could not be demonstrated in vivo, presumably because of competing systemic effects. However, chloride secretion could be induced by 300 microM IBMX in vitro. The best results were obtained under the following conditions: a long preincubation of the tissue slices in high potassium or saline buffer (preferably in high potassium buffer), oxygenated with 95% O(2) and 5% CO(2), at low temperature (4 degrees C), followed by IBMX stimulation in saline buffer at 37 degrees C. Chloride efflux was accompanied by efflux of Na and K.

1-Methyl-3-isobutylxanthine↗

PubMed2000

CA(2+) mobilization in the human submandibular duct cell line A253.

Ca(2+)mobilization induced by ATP, isoproterenol and the Ca(2+)-ATPase inhibitor thapsigargin in the human submandibular duct cell line A253 was investigated using the Ca(2+)-sensitive fluorescent indicator fura-2. ATP and isoproterenol increased cytosolic free Ca(2+)([Ca(2+)](i)) and subsequent exposure to thapsigargin after ATP or isoproterenol stimulation caused a further increase in [Ca(2+)](i). However, ATP and isoproterenol were not able to elicit a further increase in [Ca(2+)](i)after exposure of the cells to thapsigargin. Relatively few cells reacted to isoproterenol stimulation, but nearly all cells reacted to isoproterenol if ATP was added together with, or prior to isoproterenol stimulation. Moreover, the effect of ATP was potentiated by prior or simultaneous addition of isoproterenol. Furthermore, ATP decreased [Ca(2+)](i)in the presence of thapsigargin probably due to agonist-induced export of intracellular calcium. The results may suggest the existence of three thapsigargin sensitive pools; one opened by ATP acting through P(2)-purinergic receptors and IP(3), one opened by isoproterenol acting through beta2-adrenergic receptors, and a third pool not sensitive to ATP or isoproterenol.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2000

Effects of dorsal root transection on morphology and chemical composition of degenerating nerve fibers and reactive astrocytes in the dorsal funiculus.

The morphology and chemical (elemental) composition of the dorsal funiculus of the rat spinal cord were examined 1 and 7 days after unilateral transection (rhizotomy) of the L4 and L5 dorsal roots, using light and electron microscopy as well as X-ray microanalysis. Changes were observed only in the dorsal funiculus on the side of injury and included disintegration of the axonal cytoskeleton, enlargement of axonal mitochondria, and widening of the myelin lamellae of the injured axons. X-ray microanalysis demonstrated a significant increase in intraaxonal sodium at 1 day after injury. This increase was abolished at 7 days, but at this stage there was a significant lowering of potassium in axons and myelin sheaths and of phosphorus in myelin as well as a marked increase in calcium in the axoplasm of the degenerating axons. The nonneuronal cell compartment, largely composed of astrocytes, showed elevated sodium, chlorine, and calcium and lowered potassium levels. The changes in chemical composition paralleled an increase in immunoreactivity for the calcium-binding Mts1 (S100A4) protein, which is exclusively expressed by white matter astrocytes. The influx of calcium is likely to play a crucial role in the loss of axonal integrity after rhizotomy, while the alterations in potassium, and perhaps also phosphorus, may contribute to activation of the nonneuronal cells, including the up-regulation of Mts1 expression in astrocytes.

Animals↗

PubMed2000

Activation of deltaF508 CFTR in a cystic fibrosis respiratory epithelial cell line by 4-phenylbutyrate, genistein and CPX.

The cellular basis of cystic fibrosis (CF) is a defect in a cyclic adenosine monophosphate (cAMP)-activated chloride channel (CF transmembrane conductance regulator) in epithelial cells that leads to decreased chloride ion transport and impaired water transport across the cell membrane. This study investigated whether it was possible to activate the defective chloride channel in cystic fibrosis respiratory epithelial cells with 4-phenylbutyrate (4PBA), genistein and 8-cyclopentyl-1,3-dipropylxanthine (CPX). The CF bronchial epithelial cell line CFBE41o-, which expresses the deltaF508 mutation, was treated with these agents and loss of Cl-, indicating Cl- efflux, measured by X-ray microanalysis. 8-bromo-cAMP alone did not induce Cl- efflux in CFBE41o- cells, but after incubation with 4PBA a significant efflux of Cl- occurred. Stimulation of cells with a combination of genistein and cAMP also induced Cl- efflux, whereas a combination of pretreatment with 4PBA and a combined stimulation with genistein and cAMP induced an even larger Cl- efflux. Cl- efflux could also be stimulated by CPX, but this effect was not enhanced by 4PBA pretreatment. The deltaF508 mutation leads to impaired processing of the cystic fibrosis transmembrane conductance regulator. The increased efflux of chloride after 4-phenylbutyrate treatment can be explained by the fact that 4-phenylbutyrate allows the deltaF508 cystic fibrosis transmembrane conductance regulator to escape degradation and to be transported to the cell surface. Genistein and 8-cyclopentyl-1,3-dipropylxanthine act by stimulating chloride ion efflux by increasing the probability of the cystic fibrosis transmembrane conductance regulator being open. The combination of 4-phenylbutyrate and genistein may be useful in a potential pharmacological therapy for cystic fibrosis patients with the deltaF508 mutation.

Cell Line↗

PubMed2000

X-ray microanalysis of cultured keratinocytes: methodological aspects and effects of the irritant sodium lauryl sulphate on elemental composition.

Irritant substances have been shown to induce elemental changes in human and animal epidermal cells in situ. However, skin biopsies are a complicated experimental system and artefacts can be introduced by the anaesthesia necessary to take the biopsy. We therefore attempted to set up an experimental system for X-ray microanalysis (XRMA) consisting of cultured human keratinocytes. A number of methodological aspects were studied: different cell types, washing methods and different culture periods for the keratinocytes. It was also investigated whether the keratinocytes responded to exposure to sodium lauryl sulphate (SLS) with changes in their elemental composition. The concentrations of biologically important elements such as Na, Mg, P and K were different in HaCaT cells (a spontaneously immortalized non-tumorigenic cell line derived from adult human keratinocytes) compared to natural human epidermal keratinocytes. The washing procedure and time of culture influenced the intracellular elemental content, and rinsing with distilled water was preferred for further experiments. Changes in the elemental content in the HaCaT cells compatible with a pattern of cell injury followed by repair by cell proliferation were seen after treatment with 3.33 microM and 33 microM SLS. We conclude that XRMA is a useful tool for the study of functional changes in cultured keratinocytes, even though the preparation methods have to be strictly controlled. The method can conceivably be used for predicting effects of different chemicals on human skin.

Cells, Cultured↗

PubMed2000

Inflammation and structural changes in the airways of patients with atopic and nonatopic asthma. BHR Group.

The aim of the present study was to compare the cellular pattern and structural changes in the airway walls of atopic and nonatopic patients with asthma. Bronchial biopsy specimens were obtained from 13 atopic subjects with asthma, nine nonatopic patients with asthma, and seven healthy control subjects and investigated using immunohistochemical methods. The number of eosinophils increased in both asthma groups, but significantly more in the atopic group. The number of mast cells increased similarly in the two asthma groups, whereas the number of neutrophils increased only in the nonatopic asthma group. The number of T-lymphocytes (CD3-, CD4-, CD8-, CD-25-positive cells) was higher in patients with atopic asthma compared with nonatopic asthma. Interleukin-4 (IL-4) and IL-5-positive cells were more frequently found in the atopic asthma group, whereas cells staining for IL-8 were more frequent in the nonatopic group. The degree of epithelial damage was significantly higher in the atopic asthma group compared with the control subjects and the nonatopic asthmatics. The tenascin and laminin layer was significantly thicker in the atopic group compared with the group of nonatopic asthmatics. In the atopic group, there was a significant negative correlation between epithelial integrity (defined as the relative length of intact epithelium) and the eosinophil count and also between the number of CD25-positive cells and epithelial integrity. The number of mast cells correlated positively with the thickness of tenascin- and laminin-positive layers. In conclusion, we provide evidence of different patterns of involvement of inflammatory cells in atopic and nonatopic patients with asthma. There were also structural differences in the bronchial mucous membrane between atopic asthma and nonatopic asthma. This suggests that there are differences in the extent of the immunopathologic response of these clinically distinct forms of asthma.

Adolescent↗

PubMed2000

Piecemeal degranulation of peripheral blood eosinophils: a study of allergic subjects during and out of the pollen season.

The variability of serum and plasma levels of eosinophil granule proteins in different clinical conditions, interpreted as the result of different patterns of cytokine priming, suggests a selective mobilization of granule proteins. Inasmuch as piecemeal degranulation (PM) is the mechanism proposed for the differential release of eosinophil granule proteins, we decided to investigate whether blood eosinophils from allergic subjects show characteristics of PM during natural allergen challenge. Eosinophils from three birch-sensitive subjects were studied before and during the pollen season. Electron microscopy analysis showed that during the season, eosinophils presented morphologic features of PM. By immunogold labeling, eosinophil cationic protein (ECP) was detected not only in normal specific granules but also in the cytoplasm, in the vicinity of partially lucent specific granules. These results were confirmed by subcellular fractionation, where the amount of ECP associated with compartments containing small vesicles increased 2-fold during the pollen season. A study of the distribution of ECP, eosinophil peroxidase, and hexosaminidase in eosinophils of different densities showed that the profile of each of these proteins differed depending on cell density. All of these proteins decreased in the specific granule of hypodense cells and increased in other cell compartments. We conclude that allergen exposure causes PM of the peripheral blood eosinophils of allergic subjects, and that the density of these cells reflects the degree of degranulation. Our results provide novel information for the understanding of the selective mobilization of granule proteins into the circulation.

Blood Proteins↗

PubMed2000

Effect of progesterone and oestradiol on expression of connexin43 in cultured human myometrium cells.

In human myometrium, the formation of gap junctions at various stages of labour and in correlation with the concentration of progesterone and oestradiol in maternal blood was described previously by electron microscopy and laser confocal microscopy of immunohistochemically stained myometrial sections. The present investigation focused on the effect of continuous exposure of isolated myometrial tissue to progesterone and oestradiol on the number of gap junction plaques in human myometrium cells in vitro. The presence of gap junctions was evaluated by immunocytochemistry with antibodies against gap junction protein, connexin43 (Cx43). Human myometrial cells were isolated from biopsies obtained from term pregnant women who had an elective caesarean operation in the 37th or 40th week of pregnancy. The dispersed myometrial cells that were obtained by limited enzymatic digestion of the myometrial samples were maintained in monolayer culture for 1, 3 and 6 days in the presence of medium that contained foetal bovine serum and the steroids at different concentration. In primary culture, as well as after several passages, the characteristics of these cells were morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue. The obtained results showed that the cells in culture responded synchronously to the increased concentrations of oestradiol/progesterone mixtures. The number of gap junctions increased significantly on days 1, 3 and 6 in culture and showed positive correlation (p < 0.05) with the cell number when the concentration of oestradiol was raised to 1 microgram/mL in the progesterone ratio (1.0 microgram/0.5 microgram/mL). No significant correlation, however, in connexin43 gap junction number versus cell number was observed between the six experimental groups treated with progesterone only.

Antibodies↗

PubMed2000

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy.

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.

Animals↗

PubMed2000

Irritant-induced keratinocyte proliferation evaluated with two different methods: immunohistochemistry and X-ray microanalysis.

Keratinocyte proliferation in normal human skin was induced by application of irritants: sodium lauryl sulfate (SLS) or non-anoic acid (NAA). Each irritant was applied at three different concentrations to normal human skin in vivo under occlusion for 24 h. The irritative response was visually assessed for erythema, and the number of cycling cells was calculated from the number of Ki67 positive cells in cryosections from skin biopsies taken at 0, 24, 48, 72 and 96 h. In addition, to determine the elemental content of the epidermal cells during the response, two epidermal strata were examined by X-ray microanalysis (XRMA). Both the application of SLS and that of NAA resulted in erythema, and the number of Ki67 positive cells was increased at 48 h. XRMA revealed an initial increase in the sodium/potassium (Na/K) ratio, indicating cell membrane injury. Due to an increasing potassium (K) concentration the Na/K ratio decreased after 24 h, which is compatible with proliferation, in accordance with the Ki67 data. This was more evident in SLS-stimulated skin, where also the concentrations of magnesium (Mg) and phosphorus (P) increased, an observation commonly made in proliferating cells. It appears that application of SLS results in a period of cell damage lasting about 24-48 h, followed by a period of proliferation. The effects of NAA appear more complicated, and proliferation may take place in parallel with cell damage.

Adult↗

PubMed2000

Effects of nicotine on intestinal epithelial cells in vivo and in vitro: an X-ray microanalytical study.

It has been suggested that nicotine may have a beneficial effect on ulcerative colitis. Little is known, however, about the mechanism of such an effect. In the present study, the effects of nicotine on intestinal ion transport were investigated by X-ray microanalysis. In addition to an in vivo study, an in vitro system was established by pre-incubating pieces of small intestine in solutions of different ionic composition, in an attempt to stabilize the intracellular ionic composition. It was observed that nicotine caused a loss of Cl (indicative of net chloride efflux) in the villous epithelial cells and the cells of Brunner's glands, in vitro both in Na+ and in K+-containing medium. In in vivo experiments, exposure of mice for 10 days to nicotine in drinking water resulted in a lowering of the cellular chloride concentration in villous epithelial cells of the small intestine, cells of Brunner's glands and epithelial cells of the colon. The results may indicate a disturbance of fluid transport in the intestine by nicotine. Possible mechanisms, such as inhibition of uptake of chloride across the basolateral membrane, are discussed.

Animals↗

PubMed2000

Elemental changes in skin from patients with basal cell carcinoma.

Intracellular ionic shifts have been described in connection with normal and abnormal cell proliferation. This study was conducted to determine the elemental composition of a cell tumor classified as basal cell carcinoma in skin. The biopsies were taken from 10 patients. Control biopsies were taken from the non-affected skin from the same patients. Cryosections were cut and attached to carbon specimen holders. After freeze drying the sections were analyzed by X-ray microanalysis in a scanning microscope. The X-ray microanalytical results showed that the content of magnesium, phosphorus and potassium was significantly higher in the cancer cells than in the control cells (p < 0.05). The content of sodium, chlorine, calcium and sulfur was similar in both affected and unaffected cells. We conclude from these data that basal cell carcinoma has a characteristic elemental composition. It is possible that increased potassium and magnesium levels have a stimulatory effect on this tumor type, in contrast to other types of cancer cells where elevated sodium and chlorine and lowered potassium levels have frequently been found.

Adult↗

PubMed1999

Regulation of intracellular calcium by extracellular nucleotides in pig tracheal submucosal gland cells.

The mechanisms by which extracellular nucleotides (ATP and UTP) regulate intracellular Ca2+ in cultured pig tracheal gland cells were studied. The calcium response induced by ATP or UTP was composed of a peak response and a steady plateau. In the absence of extracellular Ca2+, the peak response of the cells to both ATP and UTP was smaller, and no subsequent plateau was observed. After treatment of the cells with pertussis toxin, the peak response to UTP was significantly smaller and no plateau was seen even in the presence of extracellular Ca2+, but pertussis toxin did not change the effect of ATP. Pretreatment with U107, a phospholipase C inhibitor, almost abolished the calcium response to both ATP and UTP. Immunocytochemistry showed that in these cells, the IP3 receptor was localized in the cytoplasm (including the endoplasmic reticulum) of the cells. Our results indicate that both release of calcium from the intracellular store and Ca2+ influx across the cell membrane contribute to the mobilization of [Ca2+]i upon stimulation with nucleotides, that ATP and UTP regulate intracellular Ca2+ predominantly via the G protein-phospholipase C-IP3 pathway, and that ATP and UTP may act via distinct subtypes of P2Y receptors.

Adenosine Triphosphate↗

PubMed1999

Eosinophils and neutrophils in biopsies from the middle ear of atopic children with otitis media with effusion.

OBJECTIVE AND DESIGN: The majority of patients with otitis media with effusion (OME) and atopy have been shown to have elevated levels of eosinophil cationic protein (ECP) in their middle ear fluid. The mechanism underlying these elevated levels of ECP is not clear. The purpose of this study was to investigate the feasibility of a quantitative determination of eosinophils and neutrophils in the middle ear lining by specific immunocytochemical markers, in order to study the extent of the involvement of these cells in patients with OME. METHODS: Bilateral middle ear biopsies from five children with persistent OME and atopy confirmed by in vitro testing were evaluated for the presence of eosinophils and neutrophils with monoclonal antibodies against specific granule proteins. Five subjects who had no signs of effusion or infection but were undergoing routine tympanoplasty for dry perforations served as controls. The biopsies were embedded in a plastic resin to improve the structural preservation of the target cells and to increase the resolution in the light microscope. Dual markers were used to determine which marker was better for eosinophils and neutrophils, respectively. The following markers were used: eosinophil cationic protein (EG2), and eosinophil peroxidase (EPO) for eosinophils and myeloperoxidase (MPO) and human neutrophil lipocalin (HNL) for neutrophils. RESULTS: Antibodies against EPO gave a more localized and intense staining than antibodies against EG2. Antibodies against HNL appear more specific to neutrophils than antibodies against MPO that also recognize monocytes. The number of cells was determined both in the tissue and in the mucus covering the epithelium. Eosinophils and neutrophils were present in the subepithelial connective tissue and in the mucus blanket in the middle ear of patients with OME in significantly higher number than in the control group. In general, there were more inflammatory cells in the mucus than in the tissue itself, but the number of inflammatory cells in the mucus showed a significant positive correlation with the number of inflammatory cells in the tissue. There was a significant positive correlation between the number of neutrophils and the number of eosinophils in the tissue as well as in the mucus, irrespective of which marker was used. CONCLUSION: The results of this study show the feasibility of using specific antibodies to identify eosinophils and neutrophils in the middle ear. The initial data suggest that atopic children with OME have higher numbers of such cells as compared to non-OME controls.

Adolescent↗

PubMed1999

Changes in calcium concentrations in subcellular compartments of rat submandibular gland acinar cells induced by cholinergic stimulation.

The effects of cholinergic and alpha-adrenergic stimulation on calcium concentrations in rat submandibular gland acinar cells were evaluated at the subcellular level by electron probe X-ray microanalysis, and the subcellular distribution of receptors for inositol 1,4,5-trisphosphate (IP(3)Rs) was investigated by electron microscopic immunohistochemistry. For measurement of calcium concentrations, experiments were carried out both in vivo and in vitro. In in vivo experiments, fragments of glands were removed and slam-frozen 3 min after intraperitoneal injection of an agonist. In in vitro experiments, the gland tissue was enzymatically dispersed, treated with an agonist in the presence or absence of extracellular Ca(2+), and slam-frozen. The frozen specimens were cut into ultrathin cryosections, which were then freeze dried. Spectra were collected from secretory granules, the perinuclear cytoplasm containing endoplasmic reticulum (ER), and the nucleus of the acinar cells. A clear decrease in calcium content in secretory granules was observed after cholinergic stimulation both in vivo and in vitro in the presence or absence of extracellular Ca(2+). In the presence of extracellular Ca(2+), cholinergic stimulation following alpha-adrenergic stimulation reduced the calcium content in secretory granules to the same extent as cholinergic stimulation alone. No significant changes in the calcium content of the perinuclear cytoplasm and the nucleus were noticed after cholinergic stimulation. alpha-adrenergic stimulation did not significantly affect the calcium concentrations in any of the three compartments studied. IP(3)Rs were localized to ER lamellae, but not to secretory granule membranes or to nuclear membranes. Our findings suggest that: (1) in rat submandibular acini, Ca(2+) can be released from secretory granules by exposure to cholinergic, but not alpha-adrenergic, stimuli, (2) there are, however, no IP(3)Rs present in the granular membrane and the trigger for the Ca(2+) release remains under study, and (3) the response to either type of stimulus does not involve changes in the Ca(2+) content of the nucleus or of the perinuclear cytoplasm/ER, despite the presence of IP(3)Rs in the latter structure. This may be the result of limitation in the technique used, which cannot measure changes in the proper compartment of the ER.

Adrenergic alpha-Agonists↗

PubMed1999

Effects of TNF-alpha, IFN-gamma and IL-beta on normal human bronchial epithelial cells.

Several diseases affecting the airways such as asthma are associated with both epithelial damage and increased levels of pro-inflammatory cytokines. To investigate the possible relation between cytokines and epithelial damage, the effects of tumour necrosis factor-alpha (TNF)-alpha, interferon gamma (IFN-gamma) and interleukin-1 beta (IL-1beta) on normal human bronchial epithelial cells in vitro were studied. The cells were exposed to these cytokines for 48 or 72 h, followed by morphological, immunohistochemical and metabolic studies. Transmission and scanning electron microscopical analyses demonstrated damage to the mitochondria and an increase in cell processes induced by the cytokines. The use of antibodies against desmosomal cytokeratin showed a decrease in desmosome formation in IFN-gamma-exposed cells. Decreased glucose oxidation rate and increased accumulation of nitric oxide were found in cytokine-exposed cells. Nomega-monomethyl-L-arginine (L-NMMA) reduced nitrite production. X-ray microanalysis showed an increase in the intracellular sodium/potassium ratio of the cells after exposure to cytokines, which is an indication of cell damage. The cytokines induced both necrosis and apoptosis to varying degrees. IFN-gamma and TNF-alpha generally potentiate each other's effects. In conclusion tumour necrosis factor-alpha and interferon gamma, and to a lesser extent interleukin-1beta, can cause damage to epithelial cells, which may be a factor involved in epithelial shedding in airway diseases.

Adolescent↗