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Biomedical subjects

D A Knight

Publications and source records attributed to D A Knight.

At least 19 recordsLinked to original sources
PubMed2006

Concurrent antiviral and immunosuppressive activities of leflunomide in vivo.

We previously reported that the immunosuppressive malononitrileamides leflunomide and FK778 exert antiviral activity against cytomegalovirus (CMV). In the current investigation, we tested the hypothesis that leflunomide exerts concurrent antiviral activity and immune suppression in CMV-infected cardiac allograft recipients. Lewis rats were transplanted with Brown Norway hearts and then inoculated with rat CMV. Plaque assay demonstrated that leflunomide (30 mg/kg/day) reduced viral loads by 4-6 logs, and that the reduction in viral load was unaffected by administration of uridine. Leflunomide was as effective as cyclosporine A (CsA) or tacrolimus in preservation of allograft integrity through day 28. These studies directly demonstrate the bifunctionality of leflunomide as concurrently immunosuppressive and antiviral, enhancing the promise of this agent as a clinical option for treatment of transplant recipients.

Animals↗
PubMed2001

Inhibition of angiogenesis-related endothelial activity by the experimental immunosuppressive agent leflunomide.

BACKGROUND: Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective in the prevention/control of acute and chronic rejection in animal models and currently in phase I clinical trials in human transplant recipients. This agent is also effective in the control of graft-versus-host disease, autoimmune reactions, and the growth of certain tumors. The importance of the endothelium in these disease processes led us to hypothesize that leflunomide might act directly upon the endothelial cell (EC). METHODS AND RESULTS: Assay of human EC colony formation demonstrated dose-dependent, leflunomide-mediated inhibition of EC proliferation. In addition, the organization of EC into capillary-like networks, which occurs during 18 hr of incubation on Matrigel, was progressively disrupted with increasing concentrations of leflunomide. Finally, fibrin-embedded transverse sections of murine aorta, which sprout numerous microvessels during an 11-day incubation, were inhibited from doing so in the presence of this agent. All drug concentrations used in these experiments were nontoxic and pharmacologically relevant, and none of these effects were reversible by exogenous uridine, implying that inhibition of these processes was not due to intracellular pyrimidine depletion. Furthermore, neither cyclosporine nor tacrolimus exerted inhibitory activity in any of the experiments described above. CONCLUSIONS: Data generated by these studies distinguish leflunomide among immunosuppressants as uniquely capable of inhibiting angiogenesis-related endothelial functions and suggest additional mechanisms by which this agent might intervene in the diverse array of disease processes against which it has shown therapeutic potential.

Angiogenesis Inhibitors↗
PubMed2001

Inhibition of herpes simplex virus type 1 by the experimental immunosuppressive agent leflunomide.

BACKGROUND: Despite advances in antiviral chemotherapy, herpes simplex virus type 1 (HSV-1), continues to complicate the clinical course of many allograft recipients. We have previously demonstrated that the experimental immunosuppressive agent leflunomide inhibits production of cytomegalovirus by interference with virion assembly. We test the hypothesis that this agent exerts similar antiviral activity against HSV-1. METHODS AND RESULTS: Plaque assay of virus yield from endothelial or Vero cells after inoculation with each of four clinical HSV-1 isolates demonstrated a dose-dependent reduction of virus production in the presence of pharmacologic concentrations of A77 1726, the active metabolite of leflunomide. DNA dot blot and biochemical assay of viral DNA polymerase activity indicated that A77 does not inhibit viral DNA synthesis. Rather, as visualized by transmission electron microscopic method, this agent seems to disrupt virion assembly by preventing nucleocapsid tegumentation. CONCLUSIONS: These findings, in demonstrating that leflunomide exerts antiviral activity against HSV-1 by mechanisms similar to those we have previously shown with cytomegalovirus, imply that this agent may possess broad spectrum activity against other herpesviruses.

Animals↗
PubMed2001

Role of PGE(2) in protease-activated receptor-1, -2 and -4 mediated relaxation in the mouse isolated trachea.

1. The potential mediator role of the prostanoid PGE(2) in airway smooth muscle relaxations induced by peptidic and proteolytic activators of PAR-1, PAR-2, PAR-3 and PAR-4 was investigated in carbachol-precontracted mouse isolated tracheal segments. 2. The tethered ligand domain sequences of murine PAR-1 (SFFLRN-NH(2)), PAR-2 (SLIGRL-NH(2)) and PAR-4 (GYPGKF-NH(2)), but not PAR-3 (SFNGGP-NH(2)), induced smooth muscle relaxation that was abolished by the non-selective cyclo-oxygenase (COX) inhibitor, indomethacin. The relative order for mean peak relaxation was SLIGRL-NH(2)>GYPGKF-NH(2) approximately amp; SFFLRN-NH(2)>SFNGGP-NH(2). 3. SFFLRN-NH(2), SLIGRL-NH(2) and GYPGKF-NH(2), but not SFNGGP-NH(2), induced significant PGE(2) release that was abolished by indomethacin. Like that for relaxation, the relative order for mean PGE(2) release was SLIGRL-NH(2)>GYPGKF-NH(2)>SFFLRN-NH(2)>SFNGGP-NH(2). 4. In dose-response studies, SLIGRL-NH(2) induced concentration-dependent increases in PGE(2) release (EC(50)=20.4 microM) and smooth muscle relaxation (EC(50)=15.8 microM). 5. The selective COX-2 inhibitor, nimesulide, but not the COX-1 inhibitor valeryl salicylate, significantly attenuated SLIGRL-NH(2)-induced smooth muscle relaxation and PGE(2) release. 6. Exogenously applied PGE(2) induced potent smooth muscle relaxation (EC(50)=60.3 nM) that was inhibited by the mixed DP/EP(1)/EP(2) prostanoid receptor antagonist, AH6809. SLIGRL-NH(2)-induced relaxation was also significantly inhibited by AH6809. 7. In summary, the results of this study strongly suggest that PAR-mediated relaxation in murine tracheal smooth muscle is dependent on the generation of the spasmolytic prostanoid, PGE(2). PAR-stimulated PGE(2) release appears to be generated preferentially by COX-2 rather than COX-1, and induces relaxation via activation of the EP(2) receptor.

Animals↗
PubMed2001

Protease-activated receptors in human airways: upregulation of PAR-2 in respiratory epithelium from patients with asthma.

BACKGROUND: Protease-activated receptors (PARs), which are G protein-coupled receptors that are activated after proteolytic cleavage of the amino terminus of the receptor, are likely to play a major role in airway inflammation. PARs are activated by endogenous proteases, including thrombin (PAR-1, -3, and -4) and tryptase (PAR-2 and -4), both of which are present in inflamed airways. OBJECTIVE: The purpose of this study was to compare the expression and distribution of PARs in biopsy specimens obtained from asthmatic and normal subjects and to examine the effect of inhaled corticosteroids on PAR expression. METHODS: Biopsy specimens were obtained from 10 normal and 20 asthmatic patients, and sections were stained for PAR-1, -2, -3, and -4 through use of specific antibodies. Staining was scored semiquantitatively for both intensity and distribution. RESULTS: Staining for all PARs was seen on the epithelium and smooth muscle in biopsy specimens from both normal and asthmatic subjects. In the epithelium, PAR-1 and -3 staining appeared to be apically concentrated, whereas PAR-2 and -4 staining was more diffuse. In normal subjects, epithelial staining intensity of PAR-1 and -3 was significantly greater than for PAR-4 (P < .05). Staining for PAR-1, -3, and -4 in biopsy specimens from asthmatic subjects was similar to that in specimens from normal subjects, irrespective of whether the former were using inhaled corticosteroids. However, PAR-2 staining in asthmatic epithelium was significantly increased in comparison with normal epithelium. Expression of PARs in airway smooth muscle did not differ between groups. CONCLUSION: Asthma per se is associated with increased PAR-2 expression in bronchial epithelium. Importantly, staining was not influenced by inhaled corticosteroids. These results suggest that PAR-2 might be involved in airway inflammation.

Administration, Inhalation↗
PubMed2001

Lipopolysaccharide inhibits the late-phase response to allergen by altering nitric oxide synthase activity and interleukin-10.

We have previously shown that exposure of sensitized animals to lipopolysaccharide (LPS) 18 h after ovalbumin (OVA) challenge inhibits late-airway response (LAR). Using relatively selective nitric oxide synthase (NOS) inhibitors we have shown that LPS upregulates inducible NOS (iNOS) and downregulates constitutive NOS (cNOS) activity. In this study we set out to quantitate NOS isoenzyme activity in lung homogenates and to measure ex vivo interleukin (IL)-10 in tracheal explants of naive or sensitized and OVA-challenged rats exposed to LPS. iNOS activity was increased and cNOS activity reduced 6 h after LPS exposure in naive animals (n = 6, P < 0.001) and at 18 (n = 5, P < 0.001) or 24 (n = 5, P < 0.001) h after OVA challenge in sensitized animals. LPS exposure 18 h after OVA challenge in sensitized animals reversed OVA-induced changes in NOS isoenzyme activities (n = 5, P < 0.001). In naive animals IL-10 was increased 1 h after LPS exposure (n = 5, P < 0.001), peaked at 3 h (n = 9, P < 0.001), and remained elevated above baseline at 18 h (n = 11, P < 0.05). In sensitized animals, IL-10 was not increased until 18 h after OVA challenge (n = 11, P < 0.001). Due to the rapid IL-10 increase in naive animals the released IL-10 is likely to be preformed; however, in sensitized animals the results are consistent with de novo production of IL-10. In the sensitized and OVA-challenged group, exposure to LPS 18 h after OVA produced a 3-fold increase in IL-10 at 3 h after LPS exposure (n = 5, P < 0.001). The time course and kinetics of IL-10 release in those animals was similar to that seen in naive rats. These results support our previous conclusions on the basis of in vivo studies using isoenzyme inhibitors and have shown LPS to be able to reverse OVA-induced changes in NOS isoenzyme activities during an established LAR. LPS-induced release of IL-10 is thought to play an important immunomodulatory role in this model.

Administration, Inhalation↗
PubMed2001

IL-1 genotype and adult periodontitis among young New Zealanders.

Several recent studies have investigated the association between interleukin-1 genotype and periodontitis in clinical samples, where generalizability is an issue. The aim of this study was to investigate the association between adult periodontitis and IL-1 genotype in a population-based sample of 26-year-olds. Based on probing depth (PD) measurements, participants were divided into three disease groups: "Severe" (1+ teeth with 5+mm PD; N = 25), "Moderate" (2+ teeth with 4+mm PD; N = 36), and "Controls" (the remainder; N = 800). The "periodontitis-associated genotype" (PAG; Kornman et al., 1997) was present in 20.0% of the "Severe" group and in 34.8% of "Controls", whereas the IL-1A(+4845) [1,1]/IL-1B(+3953) [2,2] genotype was present in 12.0% and 0.9%, respectively. After controlling for sex, smoking status, and plaque levels, we found that those with IL-1B(+3953) [1,1]/IL-1A(+4845) [2,2] had 12.3 times the odds of being in the "Severe" group. Analysis of these data suggests that the IL-1A(+4845) [1,1]/IL-1B(+3953) [2,2] genotype is associated with periodontal disease in this young population. Future periodontal data collections as this cohort ages are required to confirm the predictive value of that genotype.

Adult↗
PubMed2001

Regulation of human lung fibroblast phenotype and function by vitronectin and vitronectin integrins.

Myofibroblasts, characterised by high expression of alpha-smooth muscle actin (alpha-SMA), are important and transient cells in normal wound healing but are found in increased number in various pathological conditions of the lung including asthma and pulmonary fibrosis. The mechanisms that regulate the myofibroblast phenotype are unknown but are likely to involve signals from the extracellular matrix transmitted via specific integrins. Vitronectin is a glycoprotein released during inflammation and has been shown to regulate the phenotype of vascular smooth muscle cells via alpha v and beta 1 integrins. In the current study we have examined whether vitronectin influences the phenotype and function of normal human lung fibroblasts (HFL-1). Incubation of HFL-1 cells with vitronectin induced a concentration-dependent reduction in alpha-SMA expression. By contrast, function-blocking monoclonal antibodies to the vitronectin integrins alpha v, beta 1, alpha v beta 3 and alpha v beta 5 induced the expression of alpha-SMA and its organization into stress fibers. Expression of alpha-SMA induced by all function-blocking monoclonal antibodies was abrogated by inhibition of protein kinase C and phosphatidylinositol-3 kinase, but the effects of inhibition of other signalling pathways was integrin dependent. Exposure to other extracellular matrix proteins such as fibronectin, collagen or their integrins did not influence expression of alpha-SMA. The expression and organization of alpha-SMA induced by exposure to function-blocking antibodies was translated into an augmented capacity of HFL-1 cells to contract fibroblast populated collagen gels. By contrast, contraction of collagen gels following incubation with vitronectin was not significantly different to control. This study has shown that vitronectin influences the phenotype and behaviour of HFL-1 cells by downregulating the expression of alpha-SMA and reducing their contractile ability. By contrast, occupancy of specific integrins by function-blocking antibodies upregulated the expression of alpha-SMA and induced the formation of functional stress fibers capable of contracting collagen gels. These results suggest that vitronectin modulates the fibroblast-myofibroblast phenotype, implying an important role in the remodelling process during lung development or response to injury.

Actins↗
PubMed2000

Cytomegalovirus induces sialyl Lewis(x) and Lewis(x) on human endothelial cells.

BACKGROUND: Cytomegalovirus (CMV) is the primary viral cause of complications in transplant recipients. We sought to understand the mechanisms of its dissemination and induction of vascular disease, which may lead to transplant complications. Sialyl Lewis(x) (sLe(x)) and Lewis(x) (Le(x)) are known for their roles in mediating cell adhesion and as tumor-associated carbohydrate antigens. Herein we explore whether CMV induces surface expression of these important molecules in endothelial cells (EC). METHODS: Flow cytometry was used to detect surface expression of sLe(x) and Le(x) on CMV-infected human umbilical vein endothelial cells (HUVEC), with or without ultraviolet inactivation of the virus. To elucidate mechanisms of CMV-mediated induction, mRNA coding for predominant HUVEC sialyltransferases (ST) and fucosyltransferases (FT), key enzymes in sLe(x) and Le(x) synthesis, was analyzed by Northern blot. Dual immunohistochemical staining for sLe(x) and Le(x) expression of human colon and placental tissue was performed to investigate in vivo relevance. RESULTS: sLe(x) expression on CMV-infected HUVEC was strongly up-regulated by 8 days after inoculation. Le(x) expression was detectable earlier and increased steadily over time. In contrast, ultraviolet-inactivated CMV did not induce expression of these molecules. Northern blot assays demonstrated higher levels of important EC glycosyltransferases ST-IV, FT-III, and FT-IV in CMV-infected EC. Finally, high levels of sLe(x) and Le(x) were expressed in CMV-infected EC in vivo. CONCLUSIONS: Given the known biologic functions of sLe(x) and Le(x), we suggest that CMV induction of these molecules may have widespread consequences ranging from CMV dissemination to induction of CMV-associated vascular disease, including thrombosis.

Antigens, Surface↗
PubMed2000

Attenuation of cytomegalovirus-induced endothelial intercellular adhesion molecule-1 mRNA/protein expression and T lymphocyte adhesion by a 2'-O-methoxyethyl antisense oligonucleotide.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease. Although treatment of EC with ICAM-1-specific antisense oligonucleotides has been shown to attenuate ICAM-1 induction under simulated inflammatory conditions (i.e., TNF-alpha), no studies have addressed their effectiveness on virally-induced ICAM-1 expression. RESULTS: In the current investigation, we show that the progressive increase in endothelial ICAM-1 protein expression that follows inoculation with CMV correlates with a progressive accumulation of ICAM-1 mRNA. Furthermore, we demonstrate that treatment of EC with a partially 2'-O-methoxyethyl modified ICAM-1-specific antisense oligonucleotide before viral inoculation significantly reduces CMV-associated induction of ICAM-1 protein and mRNA expression. Finally, we show that antisense-mediated attenuation in ICAM-1 expression results in a significant reduction of T lymphocyte adhesion to CMV-infected EC monolayers, an interaction that has been implicated in allogeneic T lymphocyte activation, in viral transmission to transiently adherent leukocytes and subsequent hematogenous dissemination. CONCLUSIONS: These findings demonstrate for the first time that antisense oligonucleotides can effectively reverse virally-induced host cellular protein expression, specifically ICAM-1, as well as consequent T lymphocytes adhesion, thus broadening the potential clinical utility of antisense oligonucleotides.

Cell Adhesion↗
PubMed2000

Leukaemia inhibitory factor (LIF) upregulates excitatory non-adrenergic non-cholinergic and maintains cholinergic neural function in tracheal explants.

The effect of leukaemia inhibitory factor (LIF) in modulating cholinergic and sensory nerve function was examined using guinea-pig tracheal explants. Specific LIF receptors (LIFR) were immunolocalized to both cholinergic and sensory nerves. Release of SP in culture was not influenced by LIF. Similarly, maximum contraction to carbachol (C(max)) was not influenced by LIF. After 3 h, maximum (E(max)) eNANC-induced contraction in controls was 32+/-2. 5% of C(max). In LIF-treated preparations, E(max) was enhanced to 50+/-4.5% C(max) (P<0.05). Cholinergic nerve-induced contractions after 3 h incubation with LIF were similar to control. After 24 h, control E(max) was 25+/-4.5% C(max) (58% smaller than E(max) at 3 h). In contrast, in LIF-treated preparations, E(max) was 37+/-2.5% C(max), (24% smaller than at 3 h, P<0.05). This did not appear to be due to the effect of LIF on muscarinic M(2) receptor expression or function. Thus LIF appears to differentially influence the function of airway nerves and thus may provide an important link between the immune and neural systems.

Animals↗
PubMed2000

Oncostatin M synergises with house dust mite proteases to induce the production of PGE(2) from cultured lung epithelial cells.

The release of PGE(2) and nitric oxide (NO) from the respiratory epithelium may act to dampen inflammation. In other tissues, oncostatin M (OSM), a potent inducer of epithelial antiproteases, has also been shown to interact with IL-1beta to stimulate PGE(2) release. However, whether OSM interacts with pro-inflammatory cytokines and proteases in the production of anti-inflammatory eicosanoids and NO from airway epithelium is unknown. The effect of OSM and the related cytokine leukaemia inhibitory factor (LIF) on PGE(2) and NO production by the respiratory epithelial cell line, A549 in response to pro-inflammatory cytokines as well as protease-rich house dust mite (HDM) fractions and a protease-deficient rye grass pollen extract was examined by immunohistochemistry, cell culture, ELISA and enzyme-immunoassay. Cells treated with a mixture of IL-1beta, IFNgamma and LPS for 48 h produced a 9 fold increase in PGE(2) and a 3 fold increase in NO levels (both P<0.05). Both OSM and LIF were without effect. However, OSM added together with the cytokine mixture synergistically enhanced PGE(2) production (22 fold, P<0.05). OSM also synergistically enhanced PGE(2) production in response to a cysteine protease-enriched, but not serine protease-enriched HDM fraction (P<0.05). Rye grass extract, neither alone nor in combination with OSM, induced PGE(2) or NO production, although it did induce the release of GM-CSF. These observations suggest that OSM is an important co-factor in the release of PGE(2) and NO from respiratory epithelial cells and may play a role in defense against exogenous proteases such as those derived from HDM.

Animals↗
PubMed1999

Cytomegalovirus-mediated modulation of adhesion molecule expression by human arterial and microvascular endothelial cells.

BACKGROUND: Cytomegalovirus (CMV), a betaherpesvirus associated with allograft rejection, infects the endothelium, the cellular interface between allograft tissue and the host immune system. Because of recent appreciation of the phenotypic diversity of endothelial cells (EC) from different vascular compartments, controversy now exists on the universality of CMV-mediated adhesion molecule induction previously described on umbilical vein EC. Therefore, we herein extend these previous studies to arterial and microvascular EC, which represent sites of vascular rejection. METHODS: Human coronary artery, aortic, umbilical artery, and microvascular EC were mock or CMV infected and/or treated with tumor necrosis factor-a before flow cytometric and immunohistochemical analysis. RESULTS: CMV directly enhanced intercellular adhesion molecule-1 on all EC isolates but did not induce E-selectin or vascular cell adhesion molecule-1. Furthermore, CMV-infected EC were refractory to tumor necrosis factor-alpha-mediated induction of these molecules. CONCLUSION: CMV-induced modulations of adhesion molecule expression, which may affect allograft immunogenicity, seem common to all EC regardless of vascular origin.

Arteries↗
PubMed1999

Novel mechanism of inhibition of cytomegalovirus by the experimental immunosuppressive agent leflunomide.

BACKGROUND: Despite progress in antiviral chemotherapy, cytomegalovirus (CMV) remains a major cause of morbidity and mortality among pharmacologically immunosuppressed organ transplant recipients, frequently engaging the clinician in a struggle to balance graft preservation with control of CMV disease. Leflunomide, an inhibitor of protein kinase activity and pyrimidine synthesis, is an experimental immunosuppressive agent effective against acute and chronic allograft rejection in animal models. Because a number of CMV proteins are known to be phosphorylated, we tested the hypothesis that this agent might exert inhibitory activity against CMV. METHODS AND RESULTS: Plaque assays demonstrated dramatic dose-dependent attenuation of production of multiple clinical CMV isolates in leflunomide-treated human fibroblasts and endothelial cells, common targets for CMV infection in vivo. As shown by Northern blot analysis and immunohistochemical staining, leflunomide neither interferes with transcription of immediate early or late viral genes, nor with expression of corresponding proteins. CMV-specific DNA dot blots and biochemical enzyme assays indicated that, in contrast to currently approved anti-CMV drugs, leflunomide exerts no inhibitory effect on the accumulation of viral DNA in infected cells, or on viral DNA polymerase activity. Rather, as visualized by transmission electron microscopy, this agent appears to act at a late stage in virion assembly by preventing tegument acquisition by viral nucleocapsids. Finally we have demonstrated equivalent inhibitory activity of leflunomide against multi-drug-resistant CMV isolates. CONCLUSIONS: These findings imply that leflunomide, an effective immunosuppressive agent, shows potential to concurrently attenuate a major complication of immunosuppression, CMV disease, by a novel mechanism of antiviral activity.

Antiviral Agents↗
PubMed1999

Expression of FAS-independent ADP-ribosyltransferase activity by a catalytic deletion peptide of Pseudomonas aeruginosa exoenzyme S.

Earlier studies reported that Pseudomonas aeruginosa exoenzyme S (ExoS) possessed an absolute requirement for the eukaryotic protein factor activating exoenzyme S (FAS) for expressing ADP-ribosyltransferase activity. During the characterization of a serum-derived FAS-like activity, we observed the ability of a catalytic deletion peptide of ExoS (DeltaN222) to ADP-ribosylate target proteins in the absence of FAS. Characterization of the activation of DeltaN222 by FAS provided an opportunity to gain insight into the mechanism of ExoS activation by FAS. Under standard enzyme assay conditions, the initial rate of FAS-independent ADP-ribosyltransferase activity of DeltaN222 was not linear with time and rapidly approached zero. Dilution into high-ionic strength buffers stabilized DeltaN222 so it could express FAS-independent ADP-ribosyltransferase activity at a linear rate. This stabilization was a general salt effect, since dilution into a 1.0 M solution of either NaCH3COOH, NaCl, or KCl stabilized the ADP-ribosyltransferase activity of DeltaN222. Kinetic analysis in a high-ionic strength buffer showed that FAS enhanced the catalytic activity of DeltaN222 by increasing the affinity for NAD and stimulating the turnover rate. Velocity experiments indicated that the stabilization of DeltaN222 by high salt was not functionally identical to stabilization by FAS. Together, these data implicate a dual role for FAS in the allosteric activation of ExoS, involving both substrate binding and catalysis.

ADP Ribose Transferases↗
PubMed1999

Leukemia inhibitory factor is synthesized and released by human eosinophils and modulates activation state and chemotaxis.

BACKGROUND: The cytokine leukemia inhibitory factor (LIF) is known to be produced by both inflamed peripheral autonomic nerves and several cell types involved in the regulation of the immune response. We have recently demonstrated that several structural cell types in human airways produce LIF in response to inflammatory stimuli and that LIF augments contractile responses to tachykinins in airway explants. Because the eosinophil is a major effector cell in asthma and often found adjacent to the nerves, we hypothesized that eosinophils produce LIF and that LIF primes and upregulates eosinophil recruitment and function, allowing bidirectional neuroimmune interactions and augmentation of eosinophil-mediated injury. OBJECTIVE: The purpose of this study was to demonstrate that human eosinophils synthesize and release LIF, to determine the effects of LIF on eosinophil functions (ie, chemotaxis, granule protein release, expression of the activation marker CD69, and apoptosis), and to compare serum LIF levels between atopic and nonatopic individuals. METHODS: Reverse-transcription PCR, ELISA, immunocytochemistry, chemotaxis assay, and flow cytometry were used. RESULTS: Peripheral blood eosinophils express LIF and messenger RNA for LIF and LIF receptor. Serum LIF levels were higher in atopic patients with mild asthma than in nonatopic normal donors. Eosinophils from nonatopic donors were stimulated by calcium ionophore to release small amounts of LIF (from almost none to 5.3 +/- 1.8 pg/10(6) cells). Eosinophils from atopic donors showed a 10-fold increase (from 45.1 +/- 38.7 pg/106 cells to 414.5 +/- 189.9 pg/10(6) cells). Preincubation of eosinophils with LIF increased eosinophil peroxidase release 4-fold. LIF was not chemotactic for eosinophils but augmented chemotaxis mediated by substance P by 82% and by platelet-activating factor by 31%. LIF did not effect eosinophil apoptosis but increased CD69 expression. CONCLUSION: LIF has proinflammatory roles in eosinophil-dependent airway disorders.

Antigens, CD↗
PubMed1999

Expression and localization of COX-2 in human airways and cultured airway epithelial cells.

Cyclo-oxygenase is the rate-limiting enzyme in the prostanoid pathway. Although expression of the inducible isoform of cyclo-oxygenase (COX-2) is associated with cytokine-mediated inflammation, recent evidence suggests a homeostatic role for epithelial COX-2 in the gastrointestinal tract. The aim of this study was to examine the expression and localization of COX-2 in human airway epithelium both in vivo and in vitro. Human airway specimens from patients undergoing lung resection surgery for primary lung tumours (n=10) or nasal mucosal resection for non-inflammatory nasal obstruction (n=5) were examined for COX-2 expression by in situ hybridization and immunohistochemistry. COX-2 expression was also studied in two human airway epithelial cell lines (BEAS-2B and A549) using reverse transcription polymerase chain reaction and Northern and Western blot analysis. COX-2 messenger ribonucleic acid (mRNA) and protein were localized to individual columnar epithelial cells and to airway resident inflammatory cells in 9/10 lower and 5/5 upper airway specimens. Expression of COX-2 did not correlate with evidence of airway inflammation. Focal expression of COX-2 mRNA and protein was observed in bronchus-associated lymphoid tissue. Both COX-2 mRNA and protein were detected in BEAS-2B and A549 cells cultured under standard conditions. In conclusion, expression of COX-2 in human airway epithelium occurs in the upper and lower airways, is widespread in airway epithelial and airway resident inflammatory cells in the absence of overt airway inflammation, and is detectable in cultured human airway epithelial cells in the absence of inflammatory cytokine stimulation. These data suggest a potentially important homeostatic role for COX-2 in the regulation of human airway contractility, inflammation and immune responses.

Adult↗