Selective downregulation of prostaglandin E2-related pathways by the Th2 cytokine IL-13.

BACKGROUND: Levels of COX-2 and downstream products, such as prostaglandin (PG) E2, are increased in inflammatory settings after stimulation by IL-1beta, LPS, and other innate factors. Although the TH2 cytokines IL-4 and IL-13 have been reported to decrease COX-2 levels in some cell types, neither the effect of these cytokines on other PGE2-related pathways nor their effect in primary human airway epithelial cells has been evaluated. OBJECTIVE: To determine the impact of IL-13 on PGE2 pathways in primary human airway epithelial cells. METHODS: Because PGE2 has anti-inflammatory, antifibrotic, and bronchodilating properties of relevance to asthma, the effect of IL-13 (10 ng/mL for 10 days) on PGE2 pathway elements in first-passage air-liquid interface epithelial cells from 8 endobronchial brushings (5 asthmatic subjects and 3 healthy subjects) was evaluated. mRNA and protein levels for COX-1 and COX-2, membrane-bound PGE synthase 1, 15-PG dehydrogenase, and the receptors EP2 and EP4 were quantified by means of real-time PCR and Western blotting. PGE2 levels in the supernatants were measured by means of enzyme immunoassay. RESULTS: IL-13 significantly inhibited the PGE2 synthetic pathways COX-2 and PGE synthase 1 while upregulating the PGE2 metabolizing enzyme 15-PG dehydrogenase. These enzymatic changes associated and correlated with decreased supernatant PGE2 levels. Significant reductions in the mRNA for EP2 (but not EP4) were also observed. Changes in the PG pathway were both time and dose dependent (n = 3). CONCLUSION: These data suggest that IL-13 induces systematic modulation of proteins related to the production, catabolism, and function of PGE2, which might alter inflammatory and immune responses at the level of the epithelium and the submucosa below. CLINICAL IMPLICATIONS: Modulation of PGE2 pathways by IL-13 might alter inflammatory and repair processes in asthma.

Alveolar type II cells inhibit fibroblast proliferation: role of IL-1alpha.

Alveolar type II (ATII) cells inhibit fibroblast proliferation in coculture by releasing or secreting a factor(s) that stimulates fibroblast production of prostaglandin E2 (PGE2). In the present study, we sought to determine the factors released from ATII cells that stimulate PGE2 production in fibroblasts. Exogenous addition of rat IL-1alpha to cultured lung fibroblasts induced PGE2 secretion in a dose-response manner. When fibroblasts were cocultured with rat ATII cells, IL-1alpha protein was detectable in ATII cells and in the coculture medium between days 8 and 12 of culture, correlating with the highest levels of PGE2. Furthermore, under coculture conditions, IL-1alpha gene expression increased in ATII cells (but not fibroblasts) compared with either cell cultured alone. In both mixed species (human fibroblasts-rat ATII cells) and same species cocultures (rat fibroblasts and ATII cells), PGE2 secretion was inhibited by the presence of IL-1 receptor antagonist (IL-1Ra) or selective neutralizing antibody directed against rat IL-1alpha (but not IL-1beta). Conditioned media from cocultures inhibited fibroblast proliferation, and this effect was abrogated by the addition of IL-1Ra. Addition of keratinocyte growth factor (KGF) resulted in an earlier increase in PGE2 secretion and fibroblast inhibition (day 8 of coculture). This effect was inhibited by indomethacin but was not altered by IL-1Ra. We conclude that in this coculture system, IL-1alpha secretion by ATII cells is one factor that stimulates PGE2 production by lung fibroblasts, thereby inhibiting fibroblast proliferation. In addition, these studies demonstrate that KGF enhances ATII cell PGE2 production through an IL-1alpha-independent pathway.

Airway fibroblasts exhibit a synthetic phenotype in severe asthma.

BACKGROUND: Platelet-derived growth factor (PDGF) may have a significant role in airway remodeling in asthma, because it is a powerful inductor of many airway fibroblast activities such as collagen synthesis. OBJECTIVE: To determine whether PDGF is a significant contributor to airway remodeling in patients with asthma by enhancing airway fibroblast procollagen I expression. METHODS: Six normal controls without asthma, 10 subjects with mild to moderate asthma, and 5 subjects with severe asthma underwent bronchoscopy with endobronchial biopsy. Biopsies were placed in Dulbecco modified Eagle medium and fibroblasts cultured in the presence and absence of PDGF isoforms -AA, -BB, and -AB (1, 5, 10, 100 ng/mL) and insulin-like growth factor 1 (100 ng/mL). Fibroblast procollagen I and PDGF receptors (PDGFRs) alpha and beta expression were determined by ELISA. RESULTS: Platelet-derived growth factor BB significantly enhanced fibroblast procollagen I expression in patients with severe asthma compared with patients with mild/moderate asthma and normal controls. Furthermore, the baseline fibroblast expression of PDGFR-beta was significantly greater in patients with severe asthma compared with the other groups. CONCLUSION: This pilot study suggests that airway fibroblasts from patients with severe asthma exhibit a synthetic phenotype, which may be driven by the overexpression of PDGFR-beta.

Defective apoptotic cell phagocytosis attenuates prostaglandin E2 and 15-hydroxyeicosatetraenoic acid in severe asthma alveolar macrophages.

RATIONALE: Clearance of apoptotic cells is crucial to the resolution of inflammation and development of fibrosis, but the process is not well understood in normal or diseased human lungs. OBJECTIVES: To determine phagocytosis of apoptotic cells by primary human alveolar macrophages and whether defects in uptake of apoptotic cells are associated with decreases in antiinflammatory/antifibrotic mediators. METHODS: Human bronchoalveolar lavage macrophages (AMphis) from normal control subjects and subjects with mild-moderate or severe asthma were examined in vitro for phagocytosis of apoptotic human T-cell line Jurkats and secretion of inflammatory mediators. MEASUREMENTS AND MAIN RESULTS: AMphis from normal subjects and patients with mild-moderate asthma were able to phagocytose apoptotic cells in response to LPS, resulting in an induction of the antifibrotic and/or antiinflammatory eicosanoids, prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (HETE). In contrast, AMphis from patients with severe asthma had defective LPS-stimulated uptake of apoptotic cells, with associated failure to induce PGE2 and 15-HETE. In addition, LPS-stimulated basal levels of tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor were reduced in all patients with asthma, whereas PGE2 and 15-HETE were reduced only in patients with severe asthma. Dexamethasone enhanced specific uptake of apoptotic cells in all subjects, while suppressing inflammatory mediator secretion. CONCLUSIONS: A decrease in AMphis LPS-responsiveness in severe asthma is manifested by defective apoptotic cell uptake and reduces secretion of inflammatory mediators. This may contribute to the chronicity of inflammation and remodeling in lungs of patients with asthma.

Airway eicosanoids in acute severe respiratory syncytial virus bronchiolitis.

We prospectively studied the levels of eicosanoids in intubated patients with severe bronchiolitis and compared them to electively intubated non-infected infants. LeukotrieneE(4) (LTE(4)), leukotrieneB(4) (LTB(4)), and prostaglandinE(2) (PGE(2)) levels were significantly increased (P <.01) from endotracheal (ET) aspirates of infants with bronchiolitis compared with controls, as were urinary LTE(4) levels (P <.001). We conclude that eicosanoids are increased in the tracheal aspirates and urine of children with bronchiolitis.

Transforming growth factor-beta2 induces bronchial epithelial mucin expression in asthma.

The transforming growth factor (TGF)-beta family is important for tissue repair in pathological conditions including asthma. However, little is known about the impact of either TGF-beta1 or TGF-beta2 on asthmatic airway epithelial mucin expression. We evaluated bronchial epithelial TGF-beta1 and TGF-beta2 expression and their effects on mucin expression, and the role of TGF-beta1 or TGF-beta2 in interleukin (IL)-13-induced mucin expression. Epithelial TGF-beta1, TGF-beta2, and mucin expression were evaluated in endobronchial biopsies from asthmatics and normal subjects. The effects of TGF-beta1 or TGF-beta2 on mucin MUC5AC protein and mRNA expression, and the impact of IL-13 on epithelial TGF-beta1, TGF-beta2, and MUC5AC were determined in cultured bronchial epithelial cells from endobronchial brushings of both subject groups. In biopsy tissue, epithelial TGF-beta2 expression levels were higher than TGF-beta1 in both asthmatics and normals. TGF-beta2, but not TGF-beta1, was increased in asthmatics compared with normals, and significantly correlated with mucin expression. TGF-beta2, but not TGF-beta1, increased mucin expression in cultured epithelial cells from both subject groups. IL-13 increased the release of TGF-beta2, but not TGF-beta1, from epithelial cells. A neutralizing TGF-beta2 antibody partially inhibited IL-13-induced mucin expression. These data suggest that TGF-beta2 production by asthmatic bronchial epithelial cells may increase airway mucin expression. IL-13-induced mucin expression may occur in part through TGF-beta2 up-regulation.

TGF-beta and IL-13 synergistically increase eotaxin-1 production in human airway fibroblasts.

Chronic diseases may involve an "innate" response followed by an adaptive immune response, of a Th1 or Th2 variety. Little is known regarding the interactions of these responses. We hypothesized that TGF-beta1 (innate response factor associated with wound repair) in combination with IL-13 (Th2 factor) might augment inflammatory processes associated with asthma. Airway fibroblasts were cultured from asthmatic subjects and normal controls. These fibroblasts were exposed to TGF-beta1 and IL-13 alone or in combination, and eotaxin-1 expression and production were evaluated. At 48 h, eotaxin-1 production was markedly increased with the combination of TGF-beta1 and IL-13 (p < 0.0001) compared with either stimulus alone. mRNA increased slightly at 1 h with IL-13 or TGF-beta1 plus IL13, peaked, and became significantly increased over IL-13 alone at 24 h. Protein was measurable from 6 h with IL-13 and TGF-beta1 plus IL-13, but greater levels were measured over time with the combination. Actinomycin ablated the increase in mRNA and protein seen with IL-13 alone and with TGF-beta1 plus IL-13. Cycloheximide blocked the increase in mRNA at 6 h in both conditions, but also blocked the increase at 24 h with TGF-beta1 plus IL-13. STAT-6 was rapidly activated with both IL-13 and the combination, without difference. Finally, eotaxin-1-positive fibroblasts were identified in severe asthma biopsies in greater numbers than in normals. These results support the concept that interactions of innate and adaptive immune systems may be important in promoting the tissue eosinophilia of asthma, particularly in those with more severe disease.

Chemoprevention of esophageal adenocarcinoma by COX-2 inhibitors in an animal model of Barrett's esophagus.

BACKGROUND & AIMS: Carcinogenesis in Barrett's esophagus (BE) is associated with an increased expression of cyclooxygenase (COX) 2. However, there has been no direct evidence that inhibition of COX-2 prevents cancer in BE. We studied the effect of MF-Tricyclic, a selective COX-2 inhibitor, on the development of BE and adenocarcinoma in a rat model. METHODS: Four weeks after esophagojejunostomy, 105 Sprague-Dawley rats were randomized to a chow containing MF-Tricyclic or Sulindac, or a placebo. Ninety-six (92%) rats completed the study and were sacrificed at 28 +/- 2 weeks. The animals were assessed for the presence of cancer, tumor volume, BE, degree of inflammation, and COX-2 expression and activity. RESULTS: MF-Tricyclic and Sulindac reduced the relative risk of development of esophageal cancer by 55% (95% confidence interval [CI] = 43%-66%, P < 0.008) and by 79% (95% CI = 68%-87%, P < 0.001), respectively, compared with controls. No significant differences were noted in the risk of esophageal cancer between the MF-Tricyclic and the Sulindac group (P = 0.34). The median tumor volume was not significantly different among the 3 groups (P = 0.081). Moderate to severe degree of inflammation was significantly more common (P = 0.005) in the control compared with the MF-Tricyclic and the Sulindac group; however, the prevalence of BE was not significantly different between groups (P = 0.98). Rats in the control group had higher tissue PGE2 level compared with the MF-Tricyclic and Sulindac groups (P = 0.038). CONCLUSIONS: Selective and nonselective COX-2 inhibitors can inhibit inflammation, COX-2 activity, and development of adenocarcinoma induced by reflux. This provides direct evidence that COX-2 inhibitors may have chemopreventive potential in BE.

Altered expression of endothelin receptors in failing human left ventricles.

BACKGROUND: Endothelin signaling is activated in failing human hearts, and may contribute to progressive myocardial dysfunction and remodeling. However, the behavior of endothelin receptor systems (ET(A) and ET(B)) in failing human hearts is not well understood. METHODS AND RESULTS: (125)[I]-endothelin-1 binding assays conducted in the presence of a non-hydrolyzable guanine nucleotide to uncouple agonist binding demonstrated that membranes prepared from nonfailing left ventricles (LVs) exhibit a mixed pattern of ET(A) ( approximately 60%) and ET(B) ( approximately 40%) receptor protein expression. Chronic LV failure from either idiopathic dilated (IDC) or ischemic (ISC) cardiomyopathy was accompanied by a significant (P<0.001) increase in ET(A) receptor density, to approximately 80% of the total population, and a significant (P<0.02) decrease in ET(B) receptor density. Ribonuclease protection assays demonstrated an increase in ET(A) mRNA abundance in IDC and ISC LVs, and a significant (P<0.04) increase in ET(B) mRNA abundance in ISC LVs. Enzyme-linked immunoabsorbent assays demonstrated a significant increase in tissue immunoreactive endothelin-1 concentration in IDC (P=0.01) and in IDC+ISC LVs (P=0.02), but receptor subtype protein or mRNA level was not significantly correlated with tissue ET-1 across all LVs. In situ reverse-transcription polymerase chain reaction in LV sections demonstrated that in both failing and nonfailing LVs the ET(A) gene is expressed in cardiac myocytes, vascular smooth muscle and endothelium; the ET(B) gene is expressed in cardiac myocytes, fibroblasts and endothelium; and the prepro-endothelin-1 gene is expressed in myocytes and interstitial cells. CONCLUSIONS: In chronically failing human LVs, ET(A) receptor density is increased to become the dominant subtype while ET(B) receptor density is decreased. The ET(A), but not the ET(B) density change is accompanied by cognate regulation of mRNA abundance. Both receptor genes and prepro-endothelin-1 are expressed in cardiac myocytes. Finally, based on a lack of correlation with endothelin-1 tissue levels, it is unlikely that the failure-related changes in ET(A) and ET(B) receptor protein and mRNA expression result from homologous regulation by agonist exposure.