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Biomedical subjects

R Lutter

Publications and source records attributed to R Lutter.

At least 37 records · Page 2Linked to original sources
PubMed1997

Vectorial transcytosis of dimeric IgA by the Calu-3 human lung epithelial cell line: upregulation by IFN-gamma.

We have developed an in vitro airway epithelial cell model for dimeric immunoglobulin (Ig) A (dIgA) transcytosis that allows the assessment of polymeric Ig receptor (pIgR) gene expression and actual dIgA transport. Tight monolayers of human lung-derived Calu-3 adenocarcinoma cells grown on permeable membranes expressed pIgR mRNA and released more secretory component (SC; P < 0.01) and secretory IgA (sIgA; P < 0.02) into the apical medium than into the basolateral medium. Transcytosis of dIgA was not due to paracellular leakage and was inhibited to approximately 20 and 30% of control values by anti-pIgR antibodies and the competitive ligand pentameric IgM, respectively. Interferon-gamma (IFN-gamma; 200 U/ml) induced pIgR mRNA expression and increased apical release of free SC and sIgA in a dose-dependent fashion (P < 0.0001). Basolateral addition of increasing amounts of dIgA dose dependently increased apical sIgA release (P < 0.0001). These data indicate that Calu-3 monolayers are capable of translocating dIgA through the pIgR. In addition, we show the integrated stimulatory effect of IFN-gamma on pIgR mRNA and protein expression and dIgA transcytosis.

Antibodies↗
PubMed1997

Persisting Haemophilus influenzae strains induce lower levels of interleukin-6 and interleukin-8 in H292 lung epithelial cells than nonpersisting strains.

Nonencapsulated Haemophilus influenzae strains isolated from patients with chronic bronchitis can be divided into those that persist in the lower respiratory tract and those that do not. We tested the hypothesis that persisting and nonpersisting strains differ in the extent to which they activate epithelial cells to produce two potent inflammatory mediators, interleukin (IL)-6 and IL-8. A suspension of 10(7) and 10(8) colony forming units (cfu) x mL(-1) of H. influenzae, persisting and nonpersisting, induced a dose- and time-dependent production of IL-6 and IL-8 by the human pulmonary mucoepidermoid carcinoma-derived cell line H292, but levels of IL-6 were lower after exposure to persisting H. influenzae (p<0.05). IL-8 production showed a similar trend (p<0.02; analysis of variance). H. influenzae bacteria that adhered to H292 cells were equally distributed over persisting and nonpersisting isolates and induced IL-6 and IL-8 levels similar to their nonadhering counterparts. The difference between persisting and nonpersisting H. influenzae was not due to cytotoxic, antimetabolic or antiproliferative effects on H292 cells. Furthermore, pre-exposure of cells to persisting and nonpersisting isolates did not block subsequent IL-1beta-induced IL-6 production. We conclude that persisting clinical isolates induce less interleukin-6 and interleukin-8 in H292 cells than nonpersisting isolates, probably because they excrete lower amounts of a stimulus of H292 cells. The stimulus is heat stable, hydrophilic and nonproteinous and probably not lipopolysaccharide alone. These findings support the suggestion that some strains of Haemophilus influenzae that persist in the airways of patients, may do so because they induce only a weak inflammatory response.

Analysis of Variance↗
PubMed1996

Neutrophil transmigration across monolayers of endothelial cells and airway epithelial cells is regulated by different mechanisms.

Neutrophil recruitment from the blood stream into the airways is one of the important features of many inflammatory disorders in the lung. In this process, neutrophils migrate first from the blood across the ECs in the apical-to-basolateral direction into the tissues and then across airway epithelium in the basolateral-to-apical direction into the lung lumen. We investigated and compared the different mechanisms of neutrophil transmigration in vitro across monolayers of these two cell types in both directions. Neutrophil migration induced by chemoattractants was stronger across resting EC than across resting epithelial cells when both cell types were growing on top of the filters (i.e., migration in the apical-to-basolateral direction). Much higher neutrophil transepithelial migration was found in the physiological, basolateral-to-apical direction (cells hanging underneath the filters) than in the opposite direction across either resting or IL-1 beta-activated epithelial cells. In contrast, no significant difference was observed with EC under these conditions. After a 4-h IL-1 beta activation, neutrophil migration across EC was dependent on IL-8 and PAF. However, the migration across epithelial cells relied, to a greater extent, on IL-8 production, and not on PAF. The adhesion molecule ICAM-1 contributed much less to neutrophil migration across epithelium than that across endothelium. Our study provides evidence of different mechanisms of neutrophil transmigration across monolayers of EC and epithelial cells in vitro, indicating that different processes control neutrophil transmigration across EC and epithelial cells in vivo.

Bronchi↗
PubMed1996

Interleukin-8 in airway inflammation in patients with asthma and chronic obstructive pulmonary disease.

We have investigated whether IL-8 is present in airway secretions from patients with asthma and chronic obstructive pulmonary disease (COPD) to obtain information on its possible role in airway inflammation in obstructive airways disease. In the bronchoalveolar lavage fluid (BALF) from 11 clinically stable patients with asthma the levels of IL-8 were increased compared to 10 healthy subjects (median: controls 21.5 pg/ml, asthma 244 pg/ml: p < 0.005). In the patients with asthma the levels of IL-8 correlated with the percentage neutrophils in the BALF (r = 0.81; p < 0.001) and with a parameter of the permeability of the respiratory membrane, the quotient (alpha 2-macroglobulin in BALF)/(alpha 2-macroglobulin in serum) (r = 0.66; p < 0.025). In the sputum sol phase of 9 patients with symptomatic asthma the levels of IL-8 were lower than in 9 patients with COPD (asthma: 6.4 ng/ml; COPD: 16.3 ng/ml; p < 0.02) and significantly correlated with those of neutrophilic myeloperoxidase (MPO; r = 0.85; p < 0.005). The increased levels of IL-8 in the airway secretions from both patients with asthma and COPD may be markers of an ongoing inflammatory process, which is more pronounced in patients with COPD. In patients with asthma the strong correlation between the levels of IL-8 and the percentage neutrophils and/or the levels of MPO points to a role of IL-8 in the recruitment and activation of neutrophils in the airway lumen.

Adolescent↗
PubMed1996

Glucocorticosteroids affect functions of airway- and blood-derived human T-cell clones, favoring the Th1 profile through two mechanisms.

Glucocorticosteroids (GCS) are beneficial in allergic asthma. GCS therapy results in reduced mRNA expression of interleukin-4 (IL-4) and IL-5 in cells from bronchoalveolar lavage (BAL) but not of IFN-gamma. In vitro studies with blood-derived T cells, however, show inhibition of all three cytokines by GCS. We studied the effects of GCS on T cells from BAL in vitro, namely Th0-, Th1, and Th2-like clones; and we compared BAL- with blood-derived clones. Dexamethasone (DEX) inhibited the anti-CD3-induced production of IL-4, IL-5 and IFN-gamma in all 20 clones tested. IFN-gamma production was inhibited significantly less than IL-4 and IL-5. DEX enhanced the ratio IFN-gamma/IL-4 (mean +/- SEM: control, 28.7 +/- 17.6; with 10-7 M DEX, 55.0 +/- 27.5, P<0.005). Interestingly, two categories of clones were distinguished based on the effects of GCS on IL-2 production and IL-2R alpha expression and proliferation; 1) In low IL-2 producers DEX blocked IL-2 production and decreased IL-2R alpha expression and proliferation; 2) In high IL-2 producers DEX inhibited IL-2 production partially and enhanced IL-2R alpha expression and proliferation. Anti-IL-2 and anti-IL2R alpha blocked the DEX-induced increase in proliferation. High levels of added IL-2 induced the second type of response. In conclusion, the production of IL-4 and IL-5 by T-cell clones (derived either from BAL or blood) was more sensitive to inhibition by DEX than that of IFN-gamma, which may account for the therapeutic effects of glucocorticosteroids in patients with asthma. The differential effects of DEX on the proliferation of high and low IL-2 producers in vitro may implicate a selective outgrowth of Th1-like T cells in vivo in patients treated with steroids.

Antibodies↗
PubMed1996

Transmigration of human neutrophils across airway epithelial cell monolayers is preferentially in the physiologic basolateral-to-apical direction.

To study the mechanisms involved in the movement of neutrophils from the blood stream into the lung airways, we investigated human neutrophil transmigration across a monolayer of human airway epithelial cells, both in the apical-to-basolateral direction and in the more physiologic basolateral-to-apical direction. Migration of human neutrophils across monolayers of human airway epithelial H292 cell-line cells and primary bronchial epithelial cells occured most efficiently in the basolateral-to-apical direction, both after the addition of chemoattractants to resting epithelial cells and across interleukin-1beta (IL-1beta)-stimulated epithelial cells. Blocking studies with monoclonal antibodies revealed that the migration of neutrophils was mediated by the CR3 adhesion molecule (CD11b/CD18) on the neutrophils. IL-1beta-treated epithelial cells caused neutrophil movement via the secretion of chemoattractants. The most potent chemoattractant released by the epithelial cells was found to be IL-8, because the IL-1beta-induced migration was inhibited for 75 +/- 10% by the addition of an antibody against IL-8. After apical stimulation of the epithelial cells with an optimal concentration of IL-1beta, 27 +/- 4 ng/ml IL-8 was found in the supernatant at the apical side of epithelial cells. Platelet-activating factor (PAF) synthesis by the epithelial cells did not play a role in neutrophil transmigration, as was demonstrated by the lack of inhibition of this process after addition of the PAF-receptor antagonist WEB 2086. We conclude that the movement of neutrophils across airway epithelial cell monolayers occurs preferentially in the physiologic basolateral-to-apical direction, indicating that the polarity of epithelial cells is important for neutrophil transmigration.

CD18 Antigens↗
PubMed1996

Cytokine production by T-cell clones from bronchoalveolar lavage fluid of patients with asthma and healthy subjects.

Cytokines produced by T-lymphocytes play an important regulatory role in inflammation in the airways of asthmatic patients. Our aim was to analyse the cytokine production by T-cell clones from bronchoalveolar lavage fluid (BAL) of patients with allergic asthma and the cytokine production of clones from the patients' peripheral blood (PB), as well as from BAL and blood from healthy controls. In 75 randomly selected CD4+ T-cell clones, we assessed the production of interleukin (IL)-2, IL-4 and interferon-gamma (IFN-gamma). After stimulation with anti-CD3, the clones from the asthmatic patients' BAL (A-BAL) produced significantly more IL-4 and IFN-gamma (median 0.32 and 4.17 ng.mL-1, respectively) than clones from A-PB (0.11 and 1.12 ng.mL-1, respectively). No evidence was found for a dominance of a type 1 or type 2 T-helper cell (Th1- or Th2)-cytokine profile in any of the groups. In three out of nine clones tested, the stimulation with anti-CD2/CD28/phorbol myristate acetate (PMA) induced a shift of the IFN-gamma/IL-4 ratio towards a Th2-type cytokine profile. Our results suggest that the clones from the asthmatic patients' bronchoalveolar lavage were derived from a more differentiated T-cell population. In several clones, the cytokine profile was still modulated by the stimulus applied. Similarly, local conditions in the airways may be involved in directing the cytokine production of T-cells.

Adult↗
PubMed1995

Plasma protein leakage and local secretion of proteins assessed in sputum in asthma and COPD. The effect of inhaled corticosteroids.

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation with cell infiltration, increased plasma exudation and abnormal local secretion of proteins. We have analysed whether sputum differs in this respect between asthma (n = 9) and COPD (n = 9), and whether inflammatory markers in sputum are affected by treatment. In non-smoking asthma patients there was more plasma protein leakage, based on the relative coefficient of excretion Q alpha 2macroglobulin/QIgG (P = 0.03). There was less local secretion of sIgA and lactoferrin than in COPD (P < 0.05). Tryptase was slightly higher in sputum from asthma than from COPD (P < 0.05), whereas eosinophil cationic protein and myeloperoxidase were similar. After treatment with glucocorticosteroids, there was a reduction in the Q alpha 2macroglobulin/Qalbumin (P < 0.015), but no effect was seen on the levels of products from local cells. We conclude that sputum analysis is useful to study the local inflammatory process in asthma and COPD.

Administration, Inhalation↗
PubMed1995

Physical interaction between lung epithelial cells and T lymphocytes.

The present results support a role for epithelial cells in the activation of T cells in an apparent antigen-independent manner. The transient expression of CD25 indicates a short acting T cells activation. Possibly, this event primes T cells to respond swiftly upon antigen-specific stimulation or to synthesize mediators that affect the local milieu. The molecular mechanism of interaction, although not well defined possibly involves LFA3-CD2 interactions. In T cell activation, via LFA3-CD2 interaction, the density of presented LFA3 molecules is critical. With the increase in the level of expression of LFA3 by epithelial cells this critical density may have been reached. However, based on what is known about T cell activation and CD25 expression in particular it is likely that additional signals such as soluble mediators are required for T cell activation by epithelial cells. Whether this mode of activation occurs in vivo remains to be established by studying ex vivo and in situ material. Not much is known about the expression of LFA3 by epithelial cells in vivo, nor about the stimuli that induce the upregulation of LFA3. In preliminary experiments with fluorescence microscopy we found that neither TNF-alpha nor IL-1 beta induce LFA3 in the same fashion as IFN-gamma. In conclusion, T cell activation by epithelial cells could be an important feature in inflammatory and immunological processes in mucosal systems such as the bronchi and deserves further research.

Antigens, CD↗
PubMed1994

Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria.

In the crystal structure of bovine mitochondrial F1-ATPase determined at 2.8 A resolution, the three catalytic beta-subunits differ in conformation and in the bound nucleotide. The structure supports a catalytic mechanism in intact ATP synthase in which the three catalytic subunits are in different states of the catalytic cycle at any instant. Interconversion of the states may be achieved by rotation of the alpha 3 beta 3 subassembly relative to an alpha-helical domain of the gamma-subunit.

Amino Acid Sequence↗
PubMed1994

Expression of activation markers by human lung T cells after adherence to lung epithelial cells.

Both increased T cell numbers and their increased activation state have implicated an important role for T cells in chronic inflammatory reactions seen in the airways of (allergic) asthmatics. Airway epithelial cells are frequently exposed to stimuli that cause the release of mediators and the expression of cell adhesion molecules. We have examined whether human airway epithelial cells can activate lung-derived T cells. Clonal lung T cells showed an increased adherence to transformed airway epithelial cells that had been exposed previously for 2 h to human recombinant interferon-gamma (IFN-gamma; 100 U/ml). After an additional 16-24 h of culturing in the absence or presence of epithelial cells, T cells expressed increased levels of both the alpha-chain of the interleukin-2 receptor (IL-2R, CD25) and the transferrin receptor (CD71), both markers of T cell activation. T cells apparently activated by epithelial cells, however, did not produce IFN-gamma or IL-4 nor showed an increased proliferation on the addition of IL-2 (5-50 U/ml). The induced adherence to and the activation of T cells by epithelial cells is mediated largely by CD2 and its ligand lymphocyte functional antigen-3, a pathway known to up- and downregulate T cell functions.

Antigens, CD↗
PubMed1994

Analysis of plasma-protein leakage and local secretion in sputum from patients with asthma and chronic obstructive pulmonary disease.

In order to assess the usefulness of sputum analysis in studying plasma-protein exudation and local secretion of proteins in the airways, we measured specific proteins in the sputum sol phase (SSP) and sputum gel phase (SGP) from patients with stable asthma or chronic obstructive pulmonary disease (COPD). Protein levels in SSP showed relatively small variations between two subsequent visits of each patient (n = 22), as also reflected by intraclass correlation coefficients above 0.79. Protein levels differed between SSP and SGP, but inclusion of the SGP data did not affect the variation of protein levels in sputum. The degree of plasma-protein leakage was estimated from the relative coefficients of excretion (RCE) of alpha 2-macroglobulin and albumin (QA2M/QALB), and of alpha 2-macroglobulin and ceruloplasmin (QA2M/QCP), which do not depend on variable dilution of sputum. Despite the heterogeneity of the study group of 26 patients with asthma (atopic [13] smokers [13], including five patients using inhaled steroids), QA2M/QALB and QA2M/QCP correlated both with bronchial hyperreactivity (Spearman rank: r = -0.45 and r = -0.36, p < 0.05) and with blood eosinophil counts (r = 0.37 and 0.56, p < 0.05). We conclude that protein levels in SSP are relatively constant in patients with stable asthma or COPD; in patients with asthma, the plasma-protein leakage, as measured with the RCE in SSP, appears to correlate with indirect indices of airway inflammation.

Adult↗
PubMed1993

F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase.

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

Adenosine Triphosphate↗
PubMed1993

Organization and sequences of genes for the subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716.

The sequences of the genes for the nine subunits of ATP synthase in the thermophilic cyanobacterium Synechococcus 6716 have been determined. The genes were identified by comparison of the encoded proteins with sequences of ATP synthase subunits in other species, and confirmed for subunits alpha, beta, delta and epsilon, by determining their N-terminal sequences. They are arranged at three separate loci. Six of them are in one cluster in the order a: c: b': b: delta: alpha, and those for the beta and epsilon subunits form a second and separate cluster. The gene for the gamma-subunit is at a third site. As in other bacteria, the gene for subunit a is immediately preceded by a gene coding for a small hydrophobic protein of unknown function, known as uncI in Escherichia coli. The gene orders in Synechococcus 6716 are related to the orders of ATP synthase genes in the plastid genomes of higher plants, and particularly of a red alga and a diatom. The sequences of the subunits are similar to those of chloroplast ATP synthase, the alpha, beta and c subunits being particularly well conserved. Differences in the primary structures of the Synechococcus 6716 and chloroplast gamma subunits probably underlie different mechanisms of activation of ATP synthase. The nucleotide sequences that are presented also contain 12 other open reading frames. One of them encodes a protein sequence related to the E. coli DNA repair enzyme, photolyase, and another codes for a protein that contains internal repeats related to sequences in the myosin heavy chain.

Amino Acid Sequence↗
PubMed1993

Crystallization of F1-ATPase from bovine heart mitochondria.

Crystals of the F1-ATPase sector of the ATP synthase complex from bovine heart mitochondria have been grown from solutions containing polyethylene glycol 6000. The crystals diffract to 2.9 A resolution on a laboratory X-ray source. They are orthorhombic and belong to the space group P2(1)2(1)2(1). The unit cell axes are a = 285 A, b = 108 A, c = 140 A. There is one molecule of F1-ATPase in the asymmetric unit.

Animals↗
PubMed1993

Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 6.5 A resolution.

ATP synthase, the assembly which makes ATP in mitochondria, chloroplasts and bacteria, uses transmembrane proton gradients generated by respiration or photosynthesis to drive the phosphorylation of ADP. Its membrane domain is joined by a slender stalk to a peripheral catalytic domain, F1-ATPase. This domain is made of five subunits with stoichiometries of 3 alpha: 3 beta: 1 gamma: 1 delta: 1 epsilon, and in bovine mitochondria has a molecular mass of 371,000. We have determined the 3-dimensional structure of bovine mitochondrial F1-ATPase to 6.5 A resolution by X-ray crystallography. It is an approximately spherical globule 110 A in diameter, on a 40 A stem which contains two alpha-helices in a coiled-coil. This stem is presumed to be part of the stalk that connects F1 with the membrane domain in the intact ATP synthase. A pit next to the stem penetrates approximately 35 A into the F1 particle. The stem and the pit are two examples of the many asymmetric features of the structure. The central element in the asymmetry is the longer of the two alpha-helices in the stem, which extends for 90 A through the centre of the assembly and emerges on top into a dimple 15 A deep. Features with threefold and sixfold symmetry, presumed to be parts of homologous alpha and beta subunits, are arranged around the central rod and pit, but the overall structure is asymmetric. The central helix provides a possible mechanism for transmission of conformational changes induced by the proton gradient from the stalk to the catalytic sites of the enzyme.

Animals↗