Omalizumab decreased IgE-release and induced changes in cellular immunity in patients with allergic asthma.

BACKGROUND: Omalizumab, a recombinant monoclonal anti-immunoglobulin E (IgE) antibody, shows proven efficacy in the treatment of allergic diseases. A little is known about the immunological pathways affected by the decrease of circulating free IgE during omalizumab treatment. AIM OF THE STUDY: To investigate the immunological consequence of IgE withdrawal, we studied the influence of omalizumab on stimulated IgE-release of cultured peripheral blood mononuclear cells (PBMC) and on the relative number of lymphocytes in the peripheral blood (cellular immune status) in patients with allergic asthma. METHODS: Nineteen patients were enrolled and received omalizumab at a dose of at least 0.016 mg/kg/IgE (IU/ml) every 4 weeks. PBMC were isolated from peripheral blood. Cells were cultured and stimulated with IL-4 (5 ng/ml) and CD40 ligand (1 microg/ml) for 10 days. IgE release was detected in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA). Cellular immune status was investigated by fluorescence-activated cell sorting. RESULTS: Omalizumab treatment induced significant inhibition of stimulated IgE release (median 1.38-0 ng/ml vs. 1.64-2.0 ng/ml in placebo group, P<0.05). B-lymphocyte counts were also significantly lower in the omalizumab group compared with placebo after 12 weeks of treatment (median 18.2-15.6% lymphocytes vs 12.7-13.7% lymphocytes after placebo, P<0.01). There were no significant differences in the other lymphocyte subpopulations between the groups. CONCLUSIONS: These findings provide evidence of immunological influences of omalizumab treatment, leading to a downregulation of IgE secretion and decrease of lymphocyte subpopulations (B-cells) indicating their anti-inflammatory potency.

Differences in cell activation by Chlamydophila pneumoniae and Chlamydia trachomatis infection in human endothelial cells.

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.

The production, storage and release of the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 by human peripheral eosinophils in allergics and non-allergics.

BACKGROUND: Recent studies have shown that neurotrophins are produced by and can act on several immune-inflammatory cells. The origin of circulating as well as local neurotrophins is unknown. OBJECTIVES: The aim of this study was to assess whether eosinophils of allergic and non-allergic donors produce, store and release the neurotrophic factors NGF, BDNF and NT-3. METHODS: Eosinophils were purified by negative immunoselection (purity > 96%) from allergic asthmatics and non-allergic donors (25 to 53 years). The presence of mRNA for neurotrophic factors was evaluated by reverse transcription PCR. Specificity was demonstrated by cloning products and sequencing. Stored NGF, BDNF and NT-3 was demonstrated by Western-blotting and flow cytometry. Eosinophils were incubated and supernatants were collected for measurement of neurotrophic factors after cell stimulation with PAF. Neurotrophin content in eosinophil lysates was determined by ELISA. RESULTS: Eosinophils demonstrate mRNA for neurotrophins. Proteins were detectable by Western blot and FACS analysis. Neurotrophins were found in the eosinophil lysates at different amounts comparing allergic and non-allergic donors. Cell stimulation with PAF (10-8-10-5 M) after priming with GM-CSF leads to a dose-dependant release of NGF and BDNF. CONCLUSIONS: Eosinophils store, produce and release NGF, BDNF and NT-3. They are a possible source of elevated neurotrophin levels found in allergy and asthma.

Bartonella henselae induces NF-kappaB-dependent upregulation of adhesion molecules in cultured human endothelial cells: possible role of outer membrane proteins as pathogenic factors.

The endothelium is a specific target for Bartonella henselae, and endothelial cell infection represents an important step in the pathogenesis of cat scratch disease and bacillary angiomatosis. Mechanisms of Bartonella-endothelial cell interaction as well as signaling pathways involved in target cell activation were analyzed. B. henselae strain Berlin-1, isolated from bacillary angiomatosis lesions of a human immunodeficiency virus-infected patient, potently stimulated human umbilical cord vein endothelial cells (HUVEC), as determined by NF-kappaB activation and enhanced adhesion molecule expression. These effects were accompanied by increased PMN rolling on and adhesion to infected endothelial cell monolayers, as measured in a parallel-plate flow chamber assay. Monoclonal antibodies against E-selectin significantly reduced PMN rolling and adhesion. In our hands, B. henselae Berlin-1 was substantially more active than the typing strain B. henselae ATCC 49882. E-selectin and ICAM-1 upregulation occurred for up to 9 days, as verified by Northern blotting and cell surface enzyme-linked immunosorbent assay. Induction of adhesion molecules was mediated via NF-kappaB activation and could be blocked by a specific NF-kappaB inhibitor. Additional studies indicated that B. henselae-induced effects did not require living bacteria or Bartonella lipopolysaccharides. Exposure of HUVEC to purified B. henselae outer membrane proteins (OMPs), however, reproduced all aspects of endothelial cell activation. In conclusion, B. henselae, the causative agent of cat scratch disease and bacillary angiomatosis, infects and activates endothelial cells. B. henselae OMPs are sufficient to induce NF-kappaB activation and adhesion molecule expression followed by enhanced rolling and adhesion of leukocytes. These observations identify important new properties of B. henselae, demonstrating its capacity to initiate a cascade of events culminating in a proinflammatory phenotype of infected endothelial cells.

Pulmonary artery endothelial cells.

Endothelial cells function as a highly regulated barrier between blood and interstitium. They play a central role in the regulation of vascular permeability by controlling the passage of liquid and nutrients as well as the transit of white blood cells (1,2). The endothelium is involved in the inflammatory response by either secreting cytokines or responding to blood-derived mediators or signals from adjacent cells (3-6). In 1973, Jaffe and coworkers (7) first published the culture of endothelial cells isolated from human umbilical veins. Ryan and coworkers (8) described, in 1978, the first isolation of pulmonary artery endothelial cells. Porcine endothelial cells have been used to study endothelial permeability in vitro (2,10,9) adhesion (11), and blood coagulation (12). Since the regulation of endothelial permeability is critical for pulmonary gas exchange, the close vicinity to airway epithelial cells has drawn attention to interactions between both cell types. Here we describe the isolation and culture of pulmonary artery endothelial cells and outline the in vitro measurement of endothelial permeability.

Infection and activation of airway epithelial cells by Chlamydia pneumoniae.

The activation of primary human airway epithelial cells (HAECs) and of the bronchial epithelial cell line BEAS-2B by Chlamydia pneumoniae, an important respiratory pathogen, was characterized. A time-dependent enhanced release of interleukin (IL)-8 and prostaglandin-E(2) and an increased expression of the epithelial adhesion molecule intercellular adhesion molecule-1 (ICAM-1), followed by subsequent transepithelial migration of polymorphonuclear neutrophils (PMN), were also demonstrated. The transepithelial PMN migration could be blocked by an anti-ICAM-1 monoclonal antibody (MAb) but not by MAbs against IL-8. In addition, there was an enhanced C. pneumoniae-mediated activation of NF-kappaB within 30-60 min in HAECs and BEAS-2B, which was followed by increases in mRNA synthesis of IL-8, ICAM-1, and cyclooxygenase-2, with maximal effects occurring 2 h after infection. Thus, C. pneumoniae infects and activates HAECs and BEAS-2B and therefore may be able to trigger a cascade of pro- and anti-inflammatory reactions during chlamydial infections.

Increased interleukin-10 and macrophage inflammatory protein-1 alpha release from blood monocytes ex vivo during late-phase response to allergen in asthma.

BACKGROUND: We determined the effect of late-phase responses to allergen challenge of mildly allergic asthmatic patients on the expression and release of the cytokines IL-10 and MIP-1alpha from peripheral blood monocytes. METHODS: Sixteen mildly allergic asthmatics were exposed to increasing concentrations of allergen aerosol. Nine demonstrated an early response alone (single responders), while seven had an early followed by a late response (dual responders). Monocytes were isolated from venous blood before and 24 h after allergen challenge, and placed in short-term culture for determination of IL-10 mRNA expression by reverse-transcription polymerase chain reaction and protein release. MIP-1alpha protein release was measured by radioimmunoassay. RESULTS: IL-10 mRNA expression was significantly higher in dual responders than early responders alone, for unstimulated monocytes or for monocytes exposed to lipopolysaccharide or IL-1beta. In addition, the release of IL-10 and MIP-1alpha from monocytes of dual responders was also enhanced. CONCLUSIONS: Circulating monocytes are primed to release more of the cytokines, IL-10 and MIP-1alpha, in dual but not in single responders, at 24 h after allergen. They may contribute to allergen-induced inflammatory responses in the airways.

Rho proteins and the p38-MAPK pathway are important mediators for LPS-induced interleukin-8 expression in human endothelial cells.

Bacterial endotoxin (lipopolysaccharide, or LPS) has potent proinflammatory properties by acting on many cell types, including endothelial cells. Secretion of the CXC-chemokine interleukin-8 (IL-8) by LPS-activated endothelial cells contributes substantially to the inflammatory response. Using human umbilical vein endothelial cells (HUVECs), we analyzed the role of small GTP-binding Rho proteins and p38 mitogen-activated protein kinase (MAPK) for LPS-dependent IL-8 expression in endothelial cells. Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced tyrosine phosphorylation, nuclear factor (NF)-kappaB-dependent gene expression, IL-8 messenger RNA, and IL-8 protein accumulation but showed no effect on LPS-dependent p38 MAPK activation. Inhibition of p38 MAPK by SB 202190 also blocked LPS-induced NF-kappaB activation and IL-8 synthesis. Furthermore, selective activation of the p38 MAPK pathway by transient expression of a constitutively active form of MAPK kinase (MKK)6, the upstream activator of p38, was as effective as LPS with respect to IL-8 expression in HUVECs. In summary, our data suggest that LPS-induced NF-kappaB activation and IL-8 synthesis in HUVECs are regulated by both a Rho-dependent signaling pathway and the MKK6/p38 kinase cascade.

Increased interleukin-10 and macrophage inflammatoryprotein-1α release from blood monocytes ex vivo duringlate-phase response to allergen in asthma.

BACKGROUND: We determined the effect of late-phase responses to allergen challenge of mildly allergic asthmatic patients on the expression and release of the cytokines IL-10 and MIP-1α from peripheral blood monocytes. METHODS: Sixteen mildly allergic asthmatics were exposed to increasing concentrations of allergen aerosol. Nine demonstrated an early response alone (single responders), while seven had an early followed by a late response (dual responders). Monocytes were isolated from venous blood before and 24 h after allergen challenge, and placed in short-term culture for determination ofIL-10 mRNA expression by reverse-transcription polymerase chain reactionand protein release. MIP-1α protein release was measured byradioimmunoassay. RESULTS: IL-10 mRNA expression was significantly higher in dual responders than early responders alone, for unstimulated monocytes or for monocytes exposed to lipopolysaccharide or IL-1ß. In addition, the release of IL-10 and MIP-1α from monocytes of dual responders was also enhanced. CONCLUSIONS: Circulating monocytes are primed to release more of the cytokines, IL-10 and MIP-1α, in dual but not in single responders, at 24 h afterallergen. They may contribute to allergen-induced inflammatory responsesin the airways.

Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae.

Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (beta1 and beta2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-kappaB activation/translocation occurred within 10-15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis.

Identification and function of cyclic nucleotide phosphodiesterase isoenzymes in airway epithelial cells.

Epithelial cells actively participate in inflammatory airway disease by liberating mediators such as arachidonate metabolites and cytokines. Inhibition of phosphodiesterases (PDEs) may be a useful anti-inflammatory approach. The PDE isoenzyme pattern and the effects of PDE inhibition on mediator generation were analyzed in primary cultures of human and porcine airway epithelial cells (AEC) and in the bronchial epithelial cell line BEAS-2B. PDE4 and PDE5 were detected in lysates of all cell types studied. In primary cultures of human AEC, the PDE4 variants PDE4A5, PDE4C1, PDE4D2, and PDE4D3 were identified by polymerase chain reaction analysis. Evidence of the recently described PDE7 was obtained by rolipram- insensitive cyclic adenosine monophosphate (cAMP) degradation, and its presence was verified by the demonstration of PDE7 messenger RNA. Primary cultures of human airway epithelium also expressed PDE1. Enhanced epithelial cAMP levels, induced by forskolin and PDE4 inhibition, increased formation of prostaglandin E2 (PGE2), but not of interleukin (IL)-8 or 15-hydroxyeicosatetraenoic acid (15-HETE) in airway epithelial cells. Increased cyclic guanosine monophosphate levels in these cells provoked by sodium nitroprusside and the PDE5 inhibitor zaprinast reduced the PGE2 synthesis, whereas 15-HETE and IL-8 formation were unchanged. The data suggest that PDE isoenzymes are important in airway inflammation and that PDE inhibitors exert anti-inflammatory effects by acting on AEC.

Repression of cyclooxygenase-2 and prostaglandin E2 release by dexamethasone occurs by transcriptional and post-transcriptional mechanisms involving loss of polyadenylated mRNA.

The two cyclooxygenase (COX) isoforms convert arachidonic acid to precursor prostaglandins (PGs). Up-regulation of COX-2 is responsible for increased PG production in inflammation and is antagonized by corticosteriods such as dexamethasone. In human pulmonary A549 cells, interleukin-1beta (IL-1beta) increases prostaglandin E2 (PGE2) synthesis via dexamethasone-sensitive induction of COX-2. Nuclear run-off assays showed that COX-2 transcription rate was repressed 25-40% by dexamethasone, while PGE2 release, COX activity, and COX-2 protein were totally repressed. At the mRNA level, complete repression of COX-2 was only observed at later (6 h) time points. Preinduced COX-2 mRNA was also potently repressed by dexamethasone, yet suppression of transcription by actinomycin D showed little effect. This dexamethasone-dependent repression involved a reduced COX-2 mRNA half-life, was blocked by actinomycin D or cycloheximide, and was antagonized by the steroid antagonist RU38486. Repression of IL-1beta-induced PGE2 release, COX activity, and COX-2 protein by actinomycin D was only effective within the first hour following IL-1beta treatment, while dexamethasone was effective when added up to 10 h later, suggesting a functional role for post-transcriptional mechanisms of repression. Following dexamethasone treatment, shortening of the average length of COX-2 mRNA poly(A) tails was observed. Finally, ligation of the COX-2 3'-UTR to a heterologous reporter failed to confer dexamethasone sensitivity. In conclusion, these data indicate a major role for post-transcriptional mechanisms in the dexamethasone-dependent repression of COX-2 that require de novo glucocorticoid receptor-dependent transcription and translation. This mechanism involves shortening of the COX-2 poly(A) tail and requires determinants other than just the 3'-UTR for specificity.

Phosphodiesterase expression in human epithelial cells.

Epithelial cells play a critical role in airway inflammation and have the capacity to produce many inflammatory mediators, including bioactive lipids and proinflammatory cytokines. Intracellular levels of cAMP and cGMP are important in the control of inflammatory cell function. These cyclic nucleotides are inactivated via a family of phosphodiesterase (PDE) enzymes, providing a possible site for drug intervention in chronic inflammatory conditions. We studied the expression of PDE activity in an epithelial cell line (A549) and in primary human airway epithelial cells (HAECs). We measured PDE function using specific inhibitors to identify the PDE families present and used RT-PCR to elucidate the expression of PDE isogenes. Both A549 cells and HAECs predominantly expressed PDE4 activity, with lesser PDE1, PDE3, and PDE5 activity. RT-PCR identified HSPDE4A5 and HSPDE4D3 together with HSPDE7. Inhibition of PDE4 and PDE3 reduced secretion by these cells. Epithelial PDE may be an important target for PDE4 inhibitors in the development of the control of asthmatic inflammation, particularly when delivered via the inhaled route.

Two distinct phospholipases C of Listeria monocytogenes induce ceramide generation, nuclear factor-kappa B activation, and E-selectin expression in human endothelial cells.

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. We recently reported that L. monocytogenes induces up-regulation of E-selectin and other endothelial adhesion molecules and subsequent polymorphonuclear leukocyte (PMN) adhesion into cultured human endothelial cells. In the present study, we characterized the mechanisms of enhanced E-selectin expression using L. monocytogenes wild type (EGD), the isogenic in-frame deletion mutants for phosphatidylcholine (PC)- and phosphatidylinositol (PI)-specific phospholipases EGD delta plcA and EGD delta plcB, as well as the nonvirulent control strain Listeria innocua. Infection of endothelial cells with EGD delta plcA or EGD delta plcB for 6 h induced, as compared with EGD wild type, intermediate levels of E-selectin mRNA and protein as well as PMN rolling and adhesion at a shear rate of 1 dyne/cm2, indicating that both bacterial phospholipases are required for a maximal effect. Similarly, ceramide content and NF-kappa B activity were increased in L. monocytogenes-exposed endothelial cells, but only to intermediate levels for PC- or PI-phospholipase C (PLC)-deficient listerial mutants. Phospholipase effects could be mimicked by exogenously added ceramides or bacterial sphingomyelinase. The data presented indicate that PI-PLC and PC-PLC are important virulence factors for L. monocytogenes infections that induce accumulation of ceramides that in turn may act as second messengers to control host cell signal-transduction pathways leading to persistent NF-kappa B activation, increased E-selectin expression, and enhanced PMN rolling/adhesion. The ability of L. monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.

Induction of phosphodiesterases 3B, 4A4, 4D1, 4D2, and 4D3 in Jurkat T-cells and in human peripheral blood T-lymphocytes by 8-bromo-cAMP and Gs-coupled receptor agonists. Potential role in beta2-adrenoreceptor desensitization.

In this study, a potential mechanism of beta2-adrenoreceptor desensitization has been explored that is based upon the enhanced degradation of cAMP by phosphodiesterase (PDE). Pretreatment of Jurkat T-cells with 8-bromo cAMP (8-Br-cAMP) or prostaglandin E2 increased PDE3 and PDE4 activity in an actinomycin D- and cycloheximide-sensitive manner. This effect was associated with increased expression of HSPDE3B, HSPDE4A4, HSPDE4D1, HSPDE4D2, and HSPDE4D3 mRNA transcripts. Western analysis reproducibly labeled a band of immunoreactivity in vehicle-treated cells that corresponded to HSPDE4A4 (125 kDa). Although the intensity of this band was unchanged in cells treated with 8-Br-cAMP, additional 68-72-kDa proteins (HSPDE4D2, HSPDE4D1) were labeled that were not detected after vehicle. Similar results were obtained with T-lymphocytes exposed to 8-Br-cAMP and fenoterol. However, in those experiments HSPDE4A4 and HSPDE4D1 appeared to be equally expressed in vehicle- and treated cells, whereas HSPDE4D2 (72 kDa) was detected only after 8-Br-cAMP. The up-regulation of PDE activity in Jurkat T-cells abolished the ability of isoproterenol to elevate cAMP, which was partially reversed by the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine, and by the PDE3 and PDE4 inhibitors, Org 9935 and rolipram, respectively. Collectively, these data suggest that chronic treatment of T-cells with cAMP-elevating agents compromises beta2-adrenoreceptor-mediated cAMP accumulation by increasing the expression of HSPDE3B and HSPDE4D gene products.

Interleukin-4 and lipopolysaccharide synergize to induce vascular cell adhesion molecule-1 expression in human lung microvascular endothelial cells.

Recent studies suggest that increased vascular cell adhesion molecule-1 (VCAM-1) expression on vascular endothelium in bronchial mucosa biopsies correlates with interleukin-4 (IL-4) levels in bronchiolar lavage fluid of allergic asthmatics. The severity of asthma in patients allergic to house dust mite has also been shown to correlate with lipopolysaccharide (LPS), rather than allergen, concentration in dust. We hypothesized that to induce effective VCAM-1 expression in human lung microvascular endothelial cells (HLMVEC), IL-4 may require the presence of a co-stimulus such as LPS. To test this hypothesis we measured, by enzyme-linked immunosorbent assay, induction of cell adhesion molecule expression on, and human eosinophil adhesion to, cultured HLMVEC monolayers pretreated with IL-4 alone or combined with LPS. IL-4 synergized with LPS to induce VCAM-1 expression at 24, 48, or 72 h, whereas IL-4 alone induced expression at 72 h only. IL-4 did not induce expression of intercellular adhesion molecule-1 or E-selectin or alter LPS-induced expression of either. Pre-exposure of HLMVEC to LPS or IL-4 (1 h), followed by IL-4 or LPS, respectively (23 h), also induced VCAM-1 expression. Eosinophil adhesion to HLMVEC monolayers treated with IL-4 and LPS together, but not alone, significantly (P < 0.001) increased from 9.6 +/- 1.5% (control) to 26.9 +/- 3.3% and was inhibited by a monoclonal antibody against the VCAM-1 ligand, very late antigen-4. Analysis of VCAM-1 mRNA revealed synergism between IL-4 and LPS which may, in part, contribute to enhanced VCAM-1 expression. These results suggest that the presence of a co-stimulus such as LPS may be necessary for IL-4 to effectively induce VCAM-1 expression in lung microvasculature.

Rho protein inhibition blocks protein kinase C translocation and activation.

Small GTP-binding proteins of the Ras and Rho family participate in various important signalling pathways. Large clostridial cytotoxins inactivate GTPases by UDP-glucosylation. Using Clostridium difficile toxin B-10463 (TcdB) for inactivation of Rho proteins (RhoA/Rac/Cdc42) and Clostridium sordellii lethal toxin-1522 (TcsL) for inactivation of Ras-proteins (Ras/Rac/Ral, Rap) the role of these GTPases in protein kinase C (PKC) stimulation was studied. Phorbol-myristate-acetate (PMA) induced a rapid PKC translocation to and activation in the particulate cell fraction as determined by PKC-activity measurements and Western blots for PKC alpha. These effects were blocked by TcdB inhibiting Rho proteins in endothelial cells, but not in TcsL-treated cells (i.e., cells without Ras activity), suggesting that Rho GTPases (RhoA and/or Cdc42) are the most likely GTP-binding proteins responsible for PKC activation. The Rho requirement for PKC activation/translocation was also verified for human epithelial cells and for lipopolysaccharide-stimulated endothelial cells. In summary, the data presented indicate that Rho protein inhibition blocked PKC translocation/activation in endothelial and epithelial cells.

Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1alpha, granulocyte-macrophage colony-stimulating factor, and interferon-gamma release from alveolar macrophages in asthma.

We determined the effect of inhaled corticosteroid, budesonide, on the release of the anti-inflammatory cytokine, interleukin-10 (IL-10), and of pro-inflammatory cytokines, macrophage inflammatory protein-1alpha (MIP-1alpha), interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF), from blood monocytes and alveolar macrophages of mild asthmatic subjects in a double-blind, cross-over, placebo-controlled study. Budesonide reduced bronchial hyperresponsiveness and improved baseline FEV1. Alveolar macrophages were obtained by bronchoalveolar lavage performed at the end of each treatment phase. IL-10 from blood monocytes was not altered, but both IL-10 mRNA and protein expression from alveolar macrophages stimulated by lipopolysaccharide and IL-1beta were increased after corticosteroid therapy. By contrast, alveolar macrophages released significantly less MIP-1alpha, IFN-gamma, and GM-CSF after steroid treatment. In comparison to alveolar macrophages from normal nonasthmatic volunteers, those from asthmatic patients released more MIP-1alpha, IFN-gamma, and GM-CSF but lower amounts of IL-10 particularly at baseline and after IL-1beta stimulation. The ability of steroids to inhibit pro-inflammatory cytokines but to enhance the anti-inflammatory cytokine such as IL-10 may contribute to their beneficial actions in asthma. Asthma is characterized by alveolar macrophages exhibiting both an enhanced capacity to release pro-inflammatory cytokines and a reduced capacity to produce IL-10.

Partition and diffusion of sodium and chloride ions in soft charged foam: the effect of external salt concentration and mechanical deformation.

The partition and diffusion characteristics of an acrylic acid/acrylamide hydrogel, copolymerized in the pores of a polyurethane foam with sodium and chloride ions, were studied by radiochemical methodologies. The hydrogel foam swells by 51%, 80%% and 260% relative to its raw state under bath salt concentrations of 2.0, 1.0, and 0.15 M, respectively. The corresponding partition coefficients are 1.13, 1.29, and 1.99 for sodium (Na+) and 0.89, 0. 85, and 0.65 for chloride (Cl-). The diffusion coefficients are independent of bath concentration and increase linearly with hydration towards their values in water. Deformation affects partition and diffusion solely by dilatation, which determines the swelling and hydration. Comparison of the hydrogel foam with cartilage and intervertebral disc shows considerable similarities and suggests that the same mechanisms control their function.

Identification of the protein kinase C isoenzymes in human lung and airways smooth muscle at the protein and mRNA level.

The protein kinase C (PKC) isoenzymes expressed by human peripheral lung and tracheal smooth muscle resected from individuals undergoing heart-lung transplantation were identified at the protein and mRNA level. Western immunoblot analyses of human lung identified multiple PKC isoenzymes that were differentially distributed between the soluble and particulate fraction. Thus, PKC alpha, PKC betaII, PKC epsilon, and PKC zeta were recovered predominantly in the soluble fraction whereas the eta isoform was membrane-associated together with trace amounts of PKC alpha and PKC epsilon. PKC beta1-like immunoreactivity was occasionally seen although the intensity of the band was uniformly weak. Immunoreactive bands corresponding to PKCs gamma, delta, or theta were never detected. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA extracted from human lung using oligonucleotide primer pairs that recognise unique sequences in each of the PKC genes amplified cDNA fragments that corresponded to the predicted sizes of PKC alpha, PKC betaI, PKC betaII, PKC epsilon, PKC zeta, and PKC eta (consistent with the expression of PKC isoenzyme protein) and, in addition, mRNA for PKC delta; PCR fragments of the expected size for the supposedly muscle-specific isoform, PKC theta, or the atypical isoenzyme, PKC lambda, were never obtained. The complement and distribution of PKC isoforms in human trachealis were similar, but not identical, to human lung. Thus, immunoreactive bands corresponding to the alpha, betaI, betaII, epsilon, and zeta isoenzymes of PKC were routinely labelled in the cytosolic fraction. In the particulate material PKC alpha, PKC epsilon, PKC alpha, PKC eta, and PKC mu were detected by immunoblotting. With the exception of PKC zeta, RT-PCR analyses confirmed the expression of the PKC isoforms detected at the protein level and, in addition, identified mRNA for PKC delta. Collectively, these data clearly demonstrate the expression of multiple PKC isoenzymes in human lung and tracheal smooth muscle, suggesting that they subserve diverse multifunctional roles in these tissues.