Biomedical subjects

Todd A Wyatt

Publications and source records attributed to Todd A Wyatt.

At least 19 recordsLinked to original sources

PubMed2006

Effects of cigarette smoke and alcohol on ciliated tracheal epithelium and inflammatory cell recruitment.

Ciliated epithelium represents the first line of host defense against lung infection. Most alcoholics smoke and are at high risk for developing lung infections. We reported that cigarette smoke activates protein kinase C (PKC) and alcohol desensitizes ciliary beat frequency (CBF) to beta-agonists in bovine bronchial epithelial cells in vitro. The combined effect of smoke and alcohol exposure on mouse ciliated tracheal epithelium has not been studied in vivo. We hypothesized that previously observed in vitro effects of smoke and alcohol exposure could be replicated in vivo. Female C57BL/6 mice were exposed to whole body cigarette smoke only, 20% alcohol ad libitum in drinking water only, or the combination of cigarette smoke plus alcohol for 6 wk. Bronchoalveolar lavage (BAL) cell populations, CBF, and airway kinase activity were assessed. Total BAL cells were decreased in animals exposed to alcohol alone and increased in animals exposed to smoke alone. Mice receiving smoke and alcohol had cell levels similar to smoke alone. Baseline CBF was not affected in any group; however, isoproterenol stimulation of CBF was blunted by alcohol exposure and actually slowed below baseline in the smoke plus alcohol group. Isoproterenol-induced PKA activity was inhibited in mice receiving alcohol independent of smoke exposure. Smoke activated PKC independent of alcohol. The isoproterenol-induced slowing below baseline of CBF after combined smoke and alcohol exposure demonstrates a novel ciliary impairment likely related to the combination of alcohol-mediated PKA desensitization and smoke-stimulated PKC activation, possibly through acetaldehyde present in the vapor phase of cigarette smoke.

Animals↗

PubMed2006

Inhibition of protein kinase C epsilon causes ciliated bovine bronchial cell detachment.

This study defines the in vitro phenomenon of ciliated bovine bronchial epithelial cell (BBEC) detachment from the basal epithelium and regulation of cilia motility mediated through protein kinase C epsilon (PKCepsilon). The authors determined the time course of activation and downregulation of PKCepsilon by the known PKC activator phorbol 12-myristate 13-acetate (PMA) and demonstrate that chemical inhibition of PKC by calphostin C or the novel PKC isoform inhibitor Ro 31-8220 induced striking detachment of ciliated BBECs from the basal cell monolayer within 1 hour, independent of apoptosis or necrotic cell death. The results of this study support a possible novel PKCepsilon-mediated signaling pathway through which ciliated cell attachment is maintained.

Animals↗

PubMed2005

Direct involvement of the isotype-specific C-terminus of beta tubulin in ciliary beating.

In previous studies in Drosophila, Nielsen et al. hypothesized that the beta tubulin C-terminal axonemal motif ;EGEFXXX', where X is an acidic amino acid, is required for ciliary function and assembly (Nielsen et al., 2001, Curr. Biol. 11, 529-533). This motif is present in some but not all mammalian beta tubulin isotypes. We therefore investigated whether this motif is important in ciliary function in mammals. In a preparation of isolated, ATP-reactivated bovine tracheal cilia, we found that monoclonal antibodies directed against the C-terminus of betaI, betaIV and betaV tubulin blocked ciliary beating in a concentration dependent manner. Antibodies against other epitopes of beta tubulin were ineffective, as were antibodies against alpha tubulin. Peptides consisting of the axonemal motif and motif-like sequences of these isotypes blocked ciliary beating. These results suggest that the axonemal motif sequences of betaI, betaIV and betaV tubulin are essential for ciliary function. Peptides consisting of corresponding C-terminal sequences in alpha tubulin isotypes were also ineffective in blocking ciliary beating, which suggests that the C-terminus of alpha tubulin is not directly involved in cilia function in mammals.

Amino Acid Sequence↗

PubMed2005

Cigarette smoke extract increases C5a receptor expression in human bronchial epithelial cells.

We have shown that exposing human bronchial epithelial cells (HBECs) to 5% cigarette smoke extract (CSE) up-regulates C5a anaphylatoxin receptor (C5aR) expression as determined by flow cytometric analysis and immunohistochemistry. In this study, we conducted whole-cell saturation studies to quantitate the receptor number. After exposing an HBEC line (BEAS-2B) to CSE, radiolabeled C5a bound saturably with Kd = 2.71 +/- 1.03 nM (n = 4) and Bmax = 15,044 +/- 5702 receptors/cells. Without 5% CSE, no C5a binding was detected. Competitive binding studies revealed two classes of sites with distinct affinities for C5a (Ki1 = 3.28 x 10(-16) M; Ki2 = 1.60 x 10(-9) M). BEAS-2Bs were transfected with wild-type (WT) or mutant dominant-negative (DN) protein kinase C-alpha (PKC-alpha) to investigate the relationship between PKC-alpha and C5aR availability and affinity. Western blot analysis revealed a 75-kDa lysate band from cells expressing WT and DN PKC-alpha, but DN cells exposed to 5% CSE had no functional PKC activity. Pretreatment with Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] (PKC-alpha inhibitor) had no effect on DN but significantly decreased WT PKC activity. Competitive binding studies conducted on either WT or DN PKC-alpha-transfected cells also revealed two classes of binding sites for C5a having different affinities. There was a significant rightward shift of the binding curve when WT cells were pretreated with Gö6976. These data suggest that C5aR is detectable on bronchial epithelial cells exposed to CSE and that exposure to CSE increases the availability of C5a binding sites. The data also indicate that PKC-alpha may play an important role in modulating C5aR binding.

Binding, Competitive↗

PubMed2005

Alcohol intake is associated with altered pulmonary function.

Little is known about the effect of moderate alcohol intake on lung function in the general population. Because moderate alcohol intake appears to reduce cardiovascular disease risk, we hypothesized that moderate alcohol intake is associated with better pulmonary function. To test this hypothesis, we examined the association between alcohol intake and pulmonary function, measured by spirometry, in a representative sample of U.S. adults who participated in the Third National Health and Nutrition Examination Survey. A stratified multistage clustered probability design was used to select a population-based sample. Data analyzed included alcohol intake, smoking status, education, body mass, sex, age, race, diabetes status, and CHF status. The Third National Health and Nutrition Examination Survey was conducted from 1988 to 1994 by the National Center for Health Statistics of the Centers for Disease Control and Prevention, Atlanta, GA. We analyzed data from 15,294 study participants who completed extensive questionnaires in the household and a comprehensive physical examination, including pulmonary function testing, either in the household or at a specially equipped mobile examination center. Low-to-moderate alcohol intake was not associated with reduced odds of obstructive lung function. In fact, increased odds for obstructive lung pattern were observed only in former heavy drinkers. In contrast, low-to-moderate alcohol intake was associated with better forced vital capacity and forced exhaled volume in 1s in the absence of obstruction, consistent with reduced odds for lung restriction. Using a logistic regression model, we found that individuals reporting alcohol consumption had a lower risk of lung restriction both before and after adjusting for confounding factors including smoking (P< or =.001). Alcohol intake-related reduced risk for restriction was associated with lower risk of CHF, diabetes, obesity, and lower markers of inflammation (white blood cell, fibrinogen, and C-reactive protein) consistent with less lung congestion, external restriction, and/or lung inflammation. Our analyses indicate that alcohol consumption, even at very modest intake levels, is associated with less lung restriction.

Adolescent↗

PubMed2005

Malondialdehyde-acetaldehyde adducts decrease bronchial epithelial wound repair.

Most people who abuse alcohol are cigarette smokers. Previously, we have shown that malondialdehyde, an inflammation product of lipid peroxidation, and acetaldehyde, a component of both ethanol metabolism and cigarette smoke, form protein adducts that stimulate protein kinase C (PKC) activation in bronchial epithelial cells. We have also shown that PKC can regulate bronchial epithelial cell wound repair. We hypothesize that bovine serum albumin adducted with malondialdehyde and acetaldehyde (BSA-MAA) decreases bronchial epithelial cell wound repair via binding to scavenger receptors on bronchial epithelial cells. To test this, confluent monolayers of bovine bronchial epithelial cells were grown in serum-free media prior to wounding the cells. Bronchial epithelial cell wound closure was inhibited in a dose-dependent manner (up to 60%) in the presence of BSA-MAA than in media treated cells (Laboratory of Human Carcinogenesis [LHC]-9-Roswell Park Memorial Institute [RPMI]). The specific scavenger receptor ligand, fucoidan, also stimulated PKC activation and decreased wound repair. Pretreatment with fucoidan blocked malondialdehyde-acetaldehyde binding to bronchial epithelial cells. When bronchial epithelial cells were preincubated with a PKC alpha inhibitor, Gö 6976, the inhibition of wound closure by fucoidan and BSA-MAA was blocked. Western blot demonstrated the presence of several scavenger receptors on bronchial epithelial cell membranes, including SRA, SRBI, SRBII, and CD36. Scavenger receptor-mediated activation of PKC alpha may function to reduce wound healing under conditions of alcohol and cigarette smoke exposure where malondialdehyde-acetaldehyde adducts may be present.

Acetaldehyde↗

PubMed2005

Ethanol treatment reduces bovine bronchial epithelial cell migration.

BACKGROUND: Chronic ethanol abuse is associated with significant lung disease. Excessive alcohol intake increases risk for a variety of respiratory tract diseases, including pneumonia and bronchitis. Damage to airway epithelium is critical to the pathogenesis of airway disorders such as chronic bronchitis and chronic obstructive pulmonary disease. The ability of the airway epithelium to repair itself is an important step in the resolution of airway inflammation and disease. Ethanol exposure is known to modulate signaling systems in bronchial epithelial cells. We hypothesize that chronic ethanol exposure down-regulates the adenosine 3':5'-cyclic monophosphate signaling cascade in airway epithelial cells, resulting in decreased epithelial cell migration and repair. METHODS: We evaluated the effect of ethanol on primary cultures of bovine bronchial epithelial cells in in vitro models of cell migration, wound repair, cell attachment, and cell spreading. RESULTS: Ethanol causes a concentration-dependent effect on closure of mechanical wounds in cell monolayers. Pretreatment of cells with 100 mm ethanol for 24 hr further slows wound closure. Ethanol pretreatment also reduced the protein kinase A response to wounding and made the cells unresponsive to stimuli of protein kinase A that accelerate wound closure. The effects of ethanol on cell migration in wound closure were confirmed in another assay of migration, the Boyden chamber cell migration assay. Prolonged treatment with ethanol also reduced other cell functions, such as spreading and attachment, which are necessary for epithelial repair. CONCLUSIONS: Ethanol modulates signaling systems that are relevant to airway injury and repair, suggesting that chronic, heavy ethanol ingestion has a detrimental impact on airway repair. Impaired response to inflammation and injury may contribute to chronic airway disease.

Adrenergic beta-Agonists↗

PubMed2005

Smoke exposure exacerbates an ethanol-induced defect in mucociliary clearance of Streptococcus pneumoniae.

BACKGROUND: Alcoholics and smokers are particularly susceptible to pulmonary infections caused by Streptococcus pneumoniae, the pneumococcus. Infection begins when pneumococci colonizing the nasopharynx are aspirated into the lower respiratory tract. The major host defense against this movement is the mucociliary clearance apparatus. Both cigarette smoke and ethanol (EtOH) exposure alter ciliary beating and protein kinase activity in the respiratory mucosa in vitro, but their effects on bacterial clearance in the intact animal have not been determined. METHODS: Male Sprague Dawley rats were exposed twice daily for 12 weeks to either the smoke generated from 30 cigarettes (smoke-exposed) or room air (sham-exposed). For the last five weeks of smoke exposure, the rats were fed Lieber-DeCarli liquid diets containing 0%, 16%, 26%, or 36% EtOH calories. The rats then were infected intranasally with S. pneumoniae, and movement of the organisms into the lower respiratory tract was quantified by plate counts of the tracheas and lungs 4 hr later. Ciliary beat frequency (CBF) analysis was performed on tracheal ring explants from each animal before and after stimulation with the beta-agonist isoproterenol, and tracheal epithelial cell protein kinase C (PKC) activity was measured. RESULTS: Ingestion of any of the EtOH-containing diets resulted in a dose-dependent increase in movement of S. pneumoniae into the rats' lungs. This EtOH-induced defect was augmented further by concurrent smoke exposure, although smoke exposure alone had little effect on S. pneumoniae movement. Smoke, but not EtOH exposure, activated tracheal epithelial cell PKC. Increased movement of organisms into lungs correlated with a decrease in CBF and loss of the ciliary response to isoproterenol. CONCLUSION: EtOH ingestion in our model facilitated movement of S. pneumoniae into rats' lungs, a phenomenon exacerbated by concurrent smoke exposure. Furthermore, the organism's movement into the lungs correlated with a blunting of the rats' ciliary response to an established stimulus. Defects in mucociliary clearance thus may be one cause of the increased risk of pneumococcal infections in people who abuse alcohol, particularly if they also smoke.

Animals↗

PubMed2005

Tumor necrosis factor-alpha hyper-responsiveness to endotoxin in whole blood is associated with chronic bronchitis in farmers.

Many farmers experience chronic bronchitis, airflow obstruction, and asthma. It is thought that these respiratory problems may be related to workplace inhalation of organic dust containing endotoxin. The purpose of this study was to determine whether whole blood cytokine responsiveness to endotoxin is associated with airflow disorders (i.e., airflow obstruction, chronic bronchitis, and doctor-diagnosed asthma). Farmers (N = 95) were recruited from a rural cohort study and completed a respiratory symptom and history questionnaire, spirometry, and blood sampling. Blood was incubated 24 hours in the presence and absence of endotoxin and supernatants were analyzed for TNF-alpha, IL-1beta, IL-6, and IL-8. Hypo- or hyper-responsiveness to endotoxin was based on whether cytokine values were in the lower or upper 10% of the group range, respectively. A significant association existed between TNF-alpha hyper-responsiveness and chronic bronchitis. These results indicate that the whole blood cytokine assay may be useful to identify individual responsiveness to endotoxin, and may provide an additional diagnostic tool to evaluate persons potentially at risk for developing chronic bronchitis following exposure to organic dust in the workplace.

Aged↗

PubMed2004

Both cAMP and cGMP are required for maximal ciliary beat stimulation in a cell-free model of bovine ciliary axonemes.

Previously, we have shown that the ATPase-dependent motion of cilia in bovine bronchial epithelial cells (BBEC) can be regulated through the cyclic nucleotides, cAMP via the cAMP-dependent protein kinase (PKA) and cGMP via the cGMP-dependent protein kinase (PKG). Both cyclic nucleotides cause an increase in cilia beat frequency (CBF). We hypothesized that cAMP and cGMP may act directly at the level of the ciliary axoneme in BBEC. To examine this, we employed a novel cell-free system utilizing detergent-extracted axonemes. Axoneme movement was whole-field analyzed digitally with the Sisson-Ammons video analysis system. A suspension of extracted axonemes remains motionless until the addition of 1 mM ATP that establishes a baseline CBF similar to that seen when analyzing intact ciliated BBEC. Adding 10 microM cAMP or 10 microM cGMP increases CBF beyond the established ATP baseline. However, the cyclic nucleotides did not stimulate CBF in the absence of ATP. Therefore, the combination of cAMP and cGMP augments ATP-driven CBF increases at the level of isolated axoneme.

Adenosine Triphosphatases↗

PubMed2004

Transforming growth factor-beta induces contraction of activated hepatic stellate cells.

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) is a cytokine produced in abundance during liver injury. Recognizing the prominent roles that hepatic stellate cells (HSCs) and TGF-beta play in portal hypertension and fibrogenesis, respectively, we sought to evaluate the effect of TGF-beta on the contractility of activated HSCs. METHODS: Spontaneous immortalized cell lines of HSC origin were used in this study. Cells were grown in three-dimensional collagen gel lattice, transferred to 60 mm dishes and exposed to varying concentrations of TGF-beta1 in serum-free medium at 37 degrees C for up to 120 h. The area of the floating gels was measured using a Fluor S-MultiImager (Biorad), the cellular smooth muscle-alpha actin (SMA) content quantified and PKC activation studies conducted. RESULTS: TGF-beta1 induced a time- and dose-dependent decrease in lattice area up to 40% of control (P<0.05) that reflects the contraction of activated HSCs. This induced contraction was associated with increases in SMA content (3-fold, P<0.05) and PKC activation (5-fold, P<0.05) in these cells. Furthermore, pre-incubating with a PKC--specific inhibitor completely abrogated the TGF-beta-induced contraction. CONCLUSIONS: TGF-beta induces contraction of activated HSCs via an increase in SMA content and a PKC--mediated pathway.

Actins↗

PubMed2004

Desensitization of PKA-stimulated ciliary beat frequency in an ethanol-fed rat model of cigarette smoke exposure.

BACKGROUND: Our previous studies have shown that the ciliary beat frequency (CBF) of cultured ciliated airway epithelial cells exposed to chronic ethanol fails to increase in response to beta-agonist stimulation. This loss of the ciliary "flight response" correlates with an ethanol-mediated desensitization of adenosine 3':5'-cyclic monophosphate-dependent protein kinase (PKA), a known regulatory component of CBF stimulation. We hypothesized that a similar ethanol-mediated desensitization of CBF would occur in vivo. METHODS: Sprague Dawley rats were fed a liquid diet containing various concentrations of ethanol for 1 or 5 weeks. Half were exposed to cigarette smoke for 12 weeks and half were sham exposed. Animals were killed and tracheal epithelial cells analyzed for CBF and PKA activity. RESULTS: Baseline CBF (approximately 6 Hz) was unchanged in tracheal epithelial cells of rats consuming diets containing 0-36% ethanol for 5 weeks. Isoproterenol stimulated CBF to 12 to 13 Hz in the tracheal epithelial cells of control rats not administered ethanol. However, isoproterenol stimulation of CBF was blunted to 7.5 Hz in rats eating a 26% ethanol diet, and there was no stimulation of CBF in rats fed a diet containing 36% ethanol. Similarly, isoproterenol stimulated a 2- to 3-fold increase in PKA activity in control rats, but this PKA response to isoproterenol was blunted in rats fed increasing concentrations of ethanol. No isoproterenol-stimulated PKA response was observed in rats fed 36% ethanol. No ethanol-induced changes in cyclic guanosine monophosphate-dependent protein kinase or protein kinase C were observed in the rats' tracheal epithelial cells. Cigarette smoke exposure slightly elevated baseline CBF and lowered the ethanol consumption level for isoproterenol-desensitization of CBF and PKA activation to 16%. No isoproterenol desensitization was observed after 1 week of alcohol feeding. Furthermore, 36% ethanol-feeding for 1 week stimulated rat tracheal CBF and PKA. CONCLUSION: These data demonstrate that in vivo administration of ethanol to rats results in decreased ciliary beating and the desensitization of PKA. This suggests a mechanism for mucociliary clearance dysfunction in alcoholics.

Animals↗

PubMed2003

Smoke and C5a induce airway epithelial intercellular adhesion molecule-1 and cell adhesion.

The human bronchial epithelial cell is one of the first cell types to be exposed to the irritants and toxins present in inhaled cigarette smoke. The ability of the bronchial epithelium to modulate inflammatory and immune events in response to cigarette smoke is important in the pathogenesis of smoke-induced airway injury. We have shown that cigarette smoke extract and the complement anaphylatoxin C5a both independently induce increased expression of intercellular adhesion molecule (ICAM)-1 on airway epithelial monolayers compared with unstimulated cells in vitro. This enhanced ICAM-1 expression is associated with a greater capacity of the airway epithelial cells to bind mononuclear cells, a process that appears to require the proinflammatory cytokine tumor necrosis factor-alpha and protein kinase C intracellular signaling. Exposure of epithelial monolayers to the combination of cigarette smoke followed by C5a results in an additive response for ICAM-1 expression and mononuclear cell adhesion compared with smoke or C5a challenge alone. Inhibiting C5a receptor expression can attenuate these responses. These findings suggest that smoke exposure in some way enhances the functional responsiveness of the C5a receptor expressed on these airway epithelial cells for subsequent C5a-mediated increases in ICAM-1 expression and mononuclear cell adhesion. Our results may help explain the initiation and propagation of inflammatory events in vivo induced by chronic airway exposure to cigarette smoke.

Antigens, CD↗

PubMed2003

Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells.

Previously, we have shown that ethanol (EtOH) stimulates a rapid increase in the ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBECs) via the activation of PKA. We have also shown that inhibitors of nitric oxide synthase block EtOH-stimulated increases in CBF. We hypothesize that EtOH acutely stimulates CBF via the activation of both PKA and PKG pathways. Using chemiluminescence detection of nitric oxide (NO), we directly measured increases in NO production in BBECs treated with 100 mmol/L of EtOH beginning at 25 minutes. Pretreatment of BBECs with guanylyl cyclase inhibitors, ODQ or LY83583, resulted in the inhibition of EtOH-stimulated CBF. Low concentrations (1 nmol/L) of cyclic nucleotide analogues do not stimulate CBF increases. However, a combination of both 1 nmol/L of 8Br-cAMP and 8Br-cGMP stimulates a significant increase over baseline CBF. This effect could be blocked by pretreating BBECs with inhibitors of either PKA or PKG. Very high concentrations of either 8Br-cAMP or 8Br-cGMP (> or =100 micromol/L) were required to cross-activate both PKA and PKG. This suggests that cross-activation of PKA by cGMP is not occurring at the concentrations (1 nmol/L) capable of stimulating CBF. 8-pCPT-cGMPS, an antagonist analogue to cGMP, blocked EtOH-stimulated PKA activity increases. These data support that EtOH-stimulated increases in CBF require the dual activation of both PKA (via cAMP) and PKG (via NO).

Animals↗

PubMed2002

Effect of malondialdehyde-acetaldehyde-protein adducts on the protein kinase C-dependent secretion of urokinase-type plasminogen activator in hepatic stellate cells.

Previous studies from our laboratory have shown that malondialdehyde-acetaldehyde-protein adducts (MAA adducts) are formed in hepatocytes of ethanol-fed rats and directly influence the hepatic stellate cells (HSCs) to induce their secretion of chemokines and to up-regulate their expression of adhesion molecules. Since protein kinase C (PKC) is known to play a major role in many diverse intracellular signal transduction processes, we investigated whether MAA adducts influence the function of HSCs via a PKC-dependent pathway. HSCs in culture were exposed to MAA adducts, and PKC activity was determined. We observed a time- and concentration-dependent activation of PKC when cultures were exposed to BSA-MAA as compared with unmodified BSA. Using PKC isoform-specific inhibitors, we also showed that BSA-MAA induces the activation of a specific isoform of PKC, PKC-alpha, in HSCs. No activation of PKC was observed when HSCs were exposed to other aldehyde adducts such as BSA-acetaldehyde or BSA-malondialdehyde, indicating that the effects of MAA adducts on HSCs were somewhat specific. We further examined whether the observed increase in PKC activation induced by MAA adducts in HSCs, in turn, causes a functional effect. We observed that BSA-MAA induces the increased secretion of urokinase-type plasminogen activator, a key component of the plasmin-generating system, and that PKC activation is necessary for this enhanced urokinase-type plasminogen activator secretion. These results indicate that MAA adducts via a PKC-mediated pathway may regulate plasmin-mediated matrix degradation in the liver, thereby contributing to the progression of hepatic fibrosis.

Acetaldehyde↗

PubMed2002

Activation of protein kinase A accelerates bovine bronchial epithelial cell migration.

Bronchial epithelial cell migration is required for the repair of damaged airway epithelium. We hypothesized that bronchial epithelial cell migration during wound repair is influenced by cAMP and the activity of its cyclic nucleotide-dependent protein kinase, protein kinase A (PKA). We found that, when confluent monolayers of bronchial epithelial cells are wounded, an increase in PKA activity occurs. Augmentation of PKA activity with a cell-permeable analog of cAMP, dibutyryl adenosine 3',5'-cyclic monophosphate, isoproterenol, or a phosphodiesterase inhibitor accelerated migration of normal bronchial epithelial cells in in vitro wound closure assays and Boyden chamber migration assays. A role for PKA activity was also confirmed with a PKA inhibitor, KT-5720, which reduced stimulated migration. Augmentation of PKA activity reduced the levels of active Rho and the formation of focal adhesions. These studies suggest that PKA activation modulates Rho activity, migration mechanisms, and thus bronchial epithelial repair mechanisms.

Animals↗

PubMed2002

PGD(2) modulates fibroblast-mediated native collagen gel contraction.

Repair of tissues is a necessary step in restoring tissue function following injury consequent to inflammation. Many inflammatory mediators are capable of modulating not only the activity of "inflammatory cells" but also of modulating functions of parenchymal cells that may contribute to repair. Disordered repair is believed to contribute to tissue dysfunction in many inflammatory diseases, including bronchial asthma. The current study evaluated the ability of prostaglandin D(2) (PGD(2)) to modulate fibroblast repair using the in vitro contraction of three-dimensional native collagen gels as a model system. PGD(2) stimulated gel contraction in a concentration- and time-dependent manner. In contrast, the PGD(2) analog BW245C inhibited contraction. Both effects were blocked by a DP-receptor blocker (AH6809). Neither TP receptor blocker SQ29548 nor protein kinase (PK) A antagonist KT5720 hand an effect on PGD(2)-stimulated contraction, suggesting action through a novel prostaglandin D receptor. PKC inhibitor calphostin-C (10(-6) M) blocked the PGD(2) stimulation of gel contraction. A calcium-independent PKC-epsilon inhibitor (Ro31-8220), but not calcium-dependent PKC-alpha and -beta inhibitors, also blocked the PGD(2) effect on contraction, implying a role for a calcium-independent pathway. This study, therefore, supports a role for PGD(2) in tissue repair and remodeling. These effects of PGD(2) appear to be mediated through receptor-signal transduction pathways different from the cAMP-PKA pathways mediating the proinflammatory activity of PGD(2), creating the possibility for selective therapeutic manipulation.

Animals↗

PubMed2002

Protein kinase C-alpha mediates cigarette smoke extract- and complement factor 5a-stimulated interleukin-8 release in human bronchial epithelial cells.

BACKGROUND: Cigarette smoke extract (CSE) activates protein kinase C (PKC) and augments complement factor 5a (C5a)-stimulated release of the proinflammatory cytokine IL-8 in human bronchial epithelial cells (HBEC). We hypothesized that PKC activation by alternative PKC activators will also mediate C5a-stimulated IL-8 release in HBEC. METHODS: HBEC were treated with phorbol myristate acetate (100 ng/mL), calcium ionophore A23187 (2 nM), or 10 nM cholesterol-3-sulfate in the presence or absence of C5a. Interleukin-8 (IL-8) release was measured by enzyme-linked immunoadsorbent assay. RESULTS: IL-8 release by PKC activators alone was significantly higher than in unstimulated cells and was further augmented in the presence of C5a. Preincubation with the PKC inhibitor calphostin C (1 microM) significantly suppressed IL-8 release in HBEC treated with CSE and C5a. Preincubation with 10 microM TMB-8 (an intracellular calcium sequester) also significantly suppressed IL-8 release in CSE- and C5a-treated HBEC, suggesting that intracellular calcium is required for CSE- and C5a-mediated IL-8 release. When HBEC were preincubated with 30 nM of the PKCbeta-specific inhibitor LY363196, CSE- and C5a-mediated IL-8 release was not inhibited. However, with higher concentrations of LY363196 (>600 nM), which exceeds the IC50 for PKCbeta greater than 100 fold, CSE- and C5a-mediated IL-8 release was significantly suppressed. Preincubation of HBEC with 100 nM of Gö 6976, a specific PKCalpha inhibitor, significantly inhibited CSE- and C5a-mediated stimulation of IL-8 release. CONCLUSIONS: Collectively, these data suggest that PKC activators in addition to CSE augment C5a-stimulated IL-8 release from HBEC and that CSE and C5a stimulate IL-8 release in HBEC by activating the calcium-dependent PKCalpha isoform.

Bronchi↗