Peripheral blood CD4+ and CD8+ T cell type 1 and type 2 cytokine production in atopic asthmatic and normal subjects.

BACKGROUND: Increased production of IL-4 and IL-5 and decreased production of IFN-gamma by CD4+ T cells has been implicated in asthma pathogenesis. However, CD8+ T cells also produce type 1 and type 2 cytokines and the relative roles of CD4+ and CD8+ T cell cytokine production in asthma have not been previously studied. OBJECTIVE: To determine the production of the type 1 and type 2 cytokines by CD4+ and CD8+ T cell subsets in asthmatic and normal subjects. METHODS: Intracellular cytokine staining for IL-4, -5, -10, -13 and IFN-gamma was analysed in peripheral blood CD4+ and CD8+ T cells from 24 atopic asthmatic and 20 normal subjects. RESULTS: Both subsets of T cells produced all cytokines studied and there were no significant differences between CD4+ and CD8+ T cells in their capacity to produce either type 1 or type 2 cytokines. There were significantly increased frequencies of IFN-gamma-positive CD4+ (13.1 +/- 2.4%, vs. 7.3 +/- 1.4%) and CD8+ (20.0 +/- 2.9%, vs. 9.6 +/- 2.1%) T cells in asthmatic subjects compared with normal subjects (P < 0.05), but not in frequencies of CD4+ or CD8+ T cells staining positively for IL-4, -5, -10 or -13. CONCLUSION: The frequencies of peripheral blood CD8+ T cells producing type 1 and type 2 cytokines are comparable with the frequencies of CD4+ T cells. There was an increased frequency of IFN-gamma producing CD4+ and CD8+ T cells in asthmatic compared with normal subjects. Further studies investigating T cells derived from the airways and investigating various stages within the disease process are required to further elucidate the importance of type 2 and type 1 T cell cytokine production in the pathogenesis of human allergic disease.

Inhibition of DNA replication and induction of S phase cell cycle arrest by G-rich oligonucleotides.

The discovery of G-rich oligonucleotides (GROs) that have non-antisense antiproliferative activity against a number of cancer cell lines has been recently described. This biological activity of GROs was found to be associated with their ability to form stable G-quartet-containing structures and their binding to a specific cellular protein, most likely nucleolin (Bates, P. J., Kahlon, J. B., Thomas, S. D., Trent, J. O., and Miller, D. M. (1999) J. Biol. Chem. 274, 26369-26377). In this report, we further investigate the novel mechanism of GRO activity by examining their effects on cell cycle progression and on nucleic acid and protein biosynthesis. Cell cycle analysis of several tumor cell lines showed that cells accumulate in S phase in response to treatment with an active GRO. Analysis of 5-bromodeoxyuridine incorporation by these cells indicated the absence of de novo DNA synthesis, suggesting an arrest of the cell cycle predominantly in S phase. At the same time point, RNA and protein synthesis were found to be ongoing, indicating that arrest of DNA replication is a primary event in GRO-mediated inhibition of proliferation. This specific blockade of DNA replication eventually resulted in altered cell morphology and induction of apoptosis. To characterize further GRO-mediated inhibition of DNA replication, we used an in vitro assay based on replication of SV40 DNA. GROs were found to be capable of inhibiting DNA replication in the in vitro assay, and this activity was correlated to their antiproliferative effects. Furthermore, the effect of GROs on DNA replication in this assay was related to their inhibition of SV40 large T antigen helicase activity. The data presented suggest that the antiproliferative activity of GROs is a direct result of their inhibition of DNA replication, which may result from modulation of a replicative helicase activity.

Rhinoviruses infect the lower airways.

Rhinoviruses are the major cause of the common cold and a trigger of acute asthma exacerbations. Whether these exacerbations result from direct infection of the lower airway or from indirect mechanisms consequent on infection of the upper airway alone is currently unknown. Lower respiratory infection was investigated in vitro by exposing primary human bronchial epithelial cells to rhinoviruses and in vivo after experimental upper respiratory infection of human volunteers. Bronchial infection was confirmed by both approaches. Furthermore, rhinoviruses induced production of interleukin-6, -8, and -16 and RANTES and were cytotoxic to cultured respiratory epithelium. This evidence strongly supports a direct lower respiratory epithelial reaction as the initial event in the induction of rhinovirus-mediated asthma exacerbations. The frequency of infection and the nature of the inflammatory response observed are similar to those of the upper respiratory tract, suggesting that rhinovirus infections may be one of the most important causes of lower in addition to upper respiratory disease.

Antiproliferative activity of G-rich oligonucleotides correlates with protein binding.

Oligonucleotides have been extensively studied as antisense or antigene agents that can potentially modulate the expression of specific genes. These strategies rely on sequence-specific hybridization of the oligonucleotide to mRNA or genomic DNA. Recently, it has become clear that oligonucleotides often have biological activities that cannot be attributed to their sequence-specific interactions with nucleic acids. Here we describe a series of guanosine-rich phosphodiester oligodeoxynucleotides that strongly inhibit proliferation in a number of human tumor cell lines. The presence of G-quartets in the active oligonucleotides is demonstrated using an UV melting technique. We show that G-rich oligonucleotides bind to a specific cellular protein and that the biological activity of the oligonucleotides correlates with binding to this protein. The G-rich oligonucleotide-binding protein was detected in both nuclear and cytoplasmic extracts and in proteins derived from the plasma membrane of cells. We present strong evidence that this protein is nucleolin, a multifunctional phosphoprotein whose levels are related to the rate of cell proliferation. Our results indicate that binding of G-rich oligonucleotides to nucleolin may be responsible for their non-sequence-specific effects. Furthermore, these oligonucleotides represent a new class of potentially therapeutic agents with a novel mechanism of action.

Low grade rhinovirus infection induces a prolonged release of IL-8 in pulmonary epithelium.

Rhinoviruses are important respiratory pathogens implicated in asthma exacerbations. The mechanisms by which rhinoviruses trigger inflammatory responses in the lower airway are poorly understood, in particular their ability to infect the lower airway. Bronchial inflammatory cell (lymphocyte and eosinophil) recruitment has been demonstrated. IL-8 is a potent proinflammatory chemokine that is chemotactic for neutrophils, lymphocytes, eosinophils, and monocytes and may be important in the pathogenesis of virus-induced asthma. Increased levels of IL-8 have been found in nasal samples in natural and experimental rhinovirus infections. In these studies we therefore examine the ability of rhinovirus to infect a transformed lower airway epithelial cell line (A549) and to induce IL-8 protein release and mRNA induction. We observed that rhinovirus type 9 is able to undergo full viral replication in A549 cells, and peak viral titers were found 24 h after inoculation. Rhinovirus infection induced a dose- and time-dependent IL-8 release up to 5 days after infection and an increase in IL-8 mRNA expression that was maximal between 3 and 24 h after infection. UV inactivation of the virus completely inhibited replication, but only reduced IL-8 protein production and mRNA induction by half, while prevention of virus-receptor binding completely inhibited virus-induced IL-8 release, suggesting that part of the observed effects was due to viral replication and part was due to virus-receptor binding. These studies demonstrate that rhinoviruses are capable of infecting a pulmonary epithelial cell line and inducing IL-8 release. These findings may be important in understanding the pathogenesis of rhinovirus-induced asthma exacerbations.

A comparison of RT-PCR, in-situ hybridisation and in-situ RT-PCR for the detection of rhinovirus infection in paraffin sections.

We describe an in-situ RT-PCR method for the amplification of rhinovirus (RV) in fixed, paraffin-embedded HeLa cells employed as a model for human respiratory epithelium. HeLa cells were infected in-vitro with inocula of rhinovirus-16 ranging from 10(2) to 10(6) 50% tissue culture infective doses (TCID50), incubated for 18 h then fixed and processed into paraffin blocks. Sections of the cell preparation were subjected to standard RT-PCR, in-situ hybridisation (ISH) or in-situ RT-PCR using specific oligonucleotide primers or probes directed against the 5' non-coding region of RV RNA. RT-PCR was found to be capable of detecting RV16 RNA in one 8 microns-thick section of cells infected with the lowest virus titre. ISH using digoxigenin labelled oligonucleotide probes located RV16 signal in the majority of HeLa cells at the highest virus titre, but in few or no cells with the lowest virus titre. In contrast, in-situ RT-PCR detected RV16 in the majority of cells infected with this amount of RV16. There was a slight loss of morphology and fine localisation associated with the in-situ thermal cycling process. However, the sensitivity of in-situ RT-PCR is comparable to standard RT-PCR and greater than ISH for the detection of RV. In-situ RT-PCR has wide applications for sensitive localization of low copy viral and RNA sequences within cells to investigate the role of viruses in a variety of clinical conditions.

Efficient triple helix formation by oligodeoxyribonucleotides containing alpha- or beta-2-amino-5-(2-deoxy-D-ribofuranosyl) pyridine residues.

Triple helices containing C+xGxC triplets are destabilised at physiological pH due to the requirement for base protonation of 2'-deoxycytidine (dC), which has a pKa of 4.3. The C nucleoside 2-amino-5-(2'-deoxy-beta-D-ribofuranosyl)pyridine (beta-AP) is structurally analogous to dC but is considerably more basic, with a pKa of 5.93. We have synthesised 5'-psoralen linked oligodeoxyribonucleotides (ODNs) containing thymidine (dT) and either beta-AP or its alpha-anomer (alpha-AP) and have assessed their ability to form triplexes with a double-stranded target derived from standard deoxynucleotides (i.e. beta-anomers). Third strand ODNs derived from dT and beta-AP were found to have considerably higher binding affinities for the target than the corresponding ODNs derived from dT and either dC or 5-methyl-2'-deoxycytidine (5-Me-dC). ODNs containing dT and alpha-AP also showed enhanced triplex formation with the duplex target and, in addition are more stable in serum-containing medium than standard oligopyrimidine-derived ODNs or ODNs derived from dT and beta-AP. Molecular modelling studies showed that an alpha-anomeric AP nucleotide can be accommodated within an otherwise beta-anomeric triplex with only minor perturbation of the triplex structure. Molecular dynamics (MD) simulations on triplexes containing either the alpha- or beta-anomer of (N1-protonated) AP showed that in both cases the base retained two standard hydrogen bonds to its associated guanine when the 'A-type' model of the triplex was used as the start-point for the simulation, but that bifurcated hydrogen bonds resulted when the alternative 'B-type' triplex model was used. The lack of a differential stability between alpha-AP- and beta-AP-containing triplexes at pH >7, predicted from the behaviour of the B-type models, suggests that the A-type models are more appropriate.

Characteristics of triplex-directed photoadduct formation by psoralen-linked oligodeoxynucleotides.

A triplex-forming oligopyrimidine has been attached at its 5'-end to a photoreactive psoralen derivative and used to target a sequence which forms part of the coding region of the human aromatase gene. The 20 base pair sequence is not a perfect triplex target since it contains three pyrimidine interruptions within the purine-rich strand. Despite this, we have detected triplex-directed photoadduct formation at pH 7.0 between the psoralen-linked oligonucleotide and a 30mer duplex representing the aromatase target. Photoadduct formation was found to be sensitive to pH, temperature, cation concentration and the base composition of the third strand. By varying the base sequence of the target duplex around the psoralen intercalation site, we have characterised the site and mode of psoralen intercalation. The attached psoralen has been found to intercalate at the triplex-duplex junction with a strong preference for one orientation. We have shown that the psoralen will bind at the junction even when there is a preferred TpA step at an adjacent site. We have also compared the binding affinity and photoreactivity of oligodeoxyribonucleotides linked to two different psoralen derivatives and found differences in the rate of crosslinking and the extent of crosslink formation. Finally, we have examined oligodeoxyribonucleotides which are attached to psoralen by polymethylene linkers of different lengths.

Inhibition of aromatase expression by a psoralen-linked triplex-forming oligonucleotide targeted to a coding sequence.

The cytochrome P450 enzyme aromatase (P450arom) is an important target in breast cancer treatment. We have designed a 20-base pyrimidine oligodeoxynucleotide (ODN) which forms a sequence-specific triple helix (triplex) with a purine-rich tract in the P450arom coding sequence. The psoralen-linked ODN (Pso20T) formed photo-induced cross-linked products with target double-stranded DNA. Cross-linked adducts formed in vitro between ODNs and P450arom expression constructs were used to transfect COS and human MCF-7 breast cancer cells. Levels of aromatase transcripts and enzyme activity were significantly lower in cultures transfected with Pso20T-treated cDNA relative to controls. Pso20T had a lesser inhibitory effect on aromatase expression from a mutant P450arom construct, consistent with predicted effects of the mutations on triplex formation. These results are compatible with triplex-mediated interruption of transcription within intact cells.

Effect of cotton, hemp, and flax dust extracts on lung permeability in the guinea pig.

Byssinosis is an occupational lung disease in textile mill workers exposed to the respirable dusts of cotton, hemp, and flax. This study investigated the influence of aqueous extracts from these dusts on overall lung permeability in the guinea pig as an index of respiratory epithelial damage. Lung permeability was assessed by absorption into blood from the lung of inhaled technetium-99m diethylenetriamine penta-acetate (Tc-DTPA) using gamma-scintigraphy. The half-life for Tc-DTPA absorption (t1/2) was significantly reduced following a 4-week inhalation treatment with cotton, hemp, or flax dust extracts when compared to saline control. There was at least a partial return to normal permeability 7 days after stopping treatment. A single inhalation of extract did not affect the t1/2, but increased the number of neutrophils in bronchoalveolar lavage fluid 24 h postexposure. Neutrophil migration into the airspaces therefore appeared to precede the increased lung permeability. Long-term exposure was not associated with respiratory epithelial shedding, suggesting that the increased permeability reflects a loss of epithelial tight junction integrity arising from repeated exposure to as yet undefined agents in these dusts.

Detection and kinetic studies of triplex formation by oligodeoxynucleotides using real-time biomolecular interaction analysis (BIA).

Real-time biomolecular interaction analysis (BIA) has been applied to triplex formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand. Sequence-specific triplex formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surface was able to capture a DNA duplex by triplex recognition. The presence of spermine increases the rate of association between the third strand and immobilised duplex, but at elevated spermine concentrations non-specific association is observed. A preliminary kinetic analysis of triplex formation at pH 5.2 by an 11mer third strand containing thymine, cytosine and uracil is reported. Values for the association and dissociation rate constants were determined to be (1.9 +/- 0.2) x 10(3) M-1 s-1 and (8.1 +/- 1.9) x 10(-5) s-1, respectively.

Discrete pathways for arachidonic acid release from tannin versus beta-glucan-stimulated rabbit alveolar macrophages.

Previously, we observed both tannin and beta-glucan to be agonists for arachidonic acid (AA) release from rabbit alveolar macrophages. Although tannin inhibited reincorporation of exogenous AA, beta-glucan had no apparent effect, suggesting separate signal transduction pathways leading to elevated AA levels. In this study alveolar macrophages were pretreated with the tyrosine phosphatase inhibitor sodium orthovanadate then stimulated with either condensed tannin or beta-glucan. Vanadate exerted opposing effects on AA release. Furthermore, vanadate reversed the ability of tannin to inhibit reacylation. Additional studies using the phospholipase A probe bis-BODIPY-C11-PC indicated that although the known phospholipase A2 activators, calcium ionophore A23187, insoluble immune complexes, and beta-glucan, generated an increase in fluorescence consistent with phospholipase A activation, tannin had no effect. These findings suggest the increase in free AA resulting from stimulation of macrophages by either tannin or beta-glucan is produced via two different mechanisms.

Comparison of neutrophil chemotactic factor release by human and rabbit alveolar macrophages in response to tannin exposure.

It is essential to determine whether the results obtained from animal models actually reflect human disease processes. Tannin is a component of cotton dust that acts as a polyclonal cell activator in vitro. Most of the effects of tannin on alveolar macrophages (AM phi) have been studied in rabbit AM phi. Therefore, we compared tannin-mediated in vitro neutrophil chemotactic factor (NCF) secretion from normal human and rabbit AM phi. For both species the NCF secretion from AM phi was dependent on tannin dose and time of exposure. The NCF released was a lipid with a molecular weight of less than 800 daltons, suggesting that it may be a metabolite of arachidonic acid. Tannin stimulation of both human and rabbit AM phi resulted in the release of 90% unmetabolized arachidonic acid derived from both phosphatidyl choline and phosphatidyl inositol membrane lipids. The NCF secreted was not leukotriene B4 or platelet-activating factor. In conclusion, tannin mediates the release of a so far undescribed NCF from resident AM phi in rabbits and human subjects that may contribute to the pathogenesis of the acute neutrophilic alveolitis associated with cotton dust inhalation. The similarity of results obtained from human and rabbit cells supports the pertinence of using rabbit cells to study tannin-mediated effects.

Calcium influx is required for tannin-mediated arachidonic acid release from alveolar macrophages.

The role of Ca2+ was investigated in the response of alveolar macrophages to cotton tannin, an agent implicated in the lung disease byssinosis in textile mill workers. A physiological concentration of extracellular Ca2+ was found to be required for tannin-mediated release of radiolabeled arachidonic acid (AA). Flow cytometry using indo 1 indicated that tannin caused a rapid and dose-dependent Ca2+ increase in macrophages that also required extracellular Ca2+. Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid virtually abolished the Ca2+ influx mediated by tannin but had little effect on intracellular Ca2+ release induced by thapsigargin, N-formylmethionyl-leucylphenylalanine, or thimerosal. A mechanism for extracellular Ca2+ influx was demonstrated by rapid Mn2+ quenching of indo 1 by tannin. Verapamil inhibited tannin-mediated Ca2+ influx and AA release, but the effective concentration was 100 microM. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid chelated all Ca2+ in the cells and effectively abolished the tannin response. Exposure to tannin was not associated with cytotoxicity, as judged by 51Cr release. The data suggest that tannin induces Ca2+ influx in alveolar macrophages, which represents an important prerequisite for a cell-signaling pathway resulting in the accumulation of free AA.

The degradation of endogenous and exogenous proteins in cultured smooth muscle cells.

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards 125I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [3H]phenylalanine, was reduced by only 10-17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (Ea = 18 kcal/mol) and short-lived proteins (Ea = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37 degrees C in contrast to the breakdown of LDL which ceased below 20 degrees C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.