Arginine deficiency augments inflammatory mediator production by airway epithelial cells in vitro.

BACKGROUND: Previously we showed that reduced availability of the essential amino acid tryptophan per se attenuates post-transcriptional control of interleukin (IL)-6 and IL-8 leading to hyperresponsive production of these inflammatory mediators by airway epithelial cells. Availability of the non-essential amino acid arginine in the inflamed airway mucosa of patients with asthma is reduced markedly, but it is not known whether this can also lead to an exaggerated production of IL-6 and IL-8. METHODS: IL-6 and IL-8 were determined by ELISA in culture supernatants of NCI-H292 airway epithelial-like cells and normal bronchial epithelial (NHBE) cells that were exposed to TNF-alpha, LPS or no stimulus, in medium with or without arginine. Arginine deficiency may also result from exposure to poly-L-arginine or major basic protein (MBP), which can block arginine uptake. Epithelial cells were exposed to these polycationic proteins and L-(14)C-arginine uptake was assessed as well as IL-6 and IL-8 production. To determine the mode of action, IL-6 and IL-8 mRNA profiles over time were assessed as were gene transcription and post-transcriptional mRNA degradation. RESULTS: For both NCI-H292 and NHBE cells, low arginine concentrations enhanced basal epithelial IL-6 and IL-8 production and synergized with TNF-alpha-induced IL-6 and IL-8 production. Poly-L-arginine enhanced the stimulus-induced IL-6 and IL-8 production, however, blocking arginine uptake and the enhanced IL-6 and IL-8 production appeared unrelated. The exaggerated IL-6 and IL-8 production due to arginine deficiency and to poly-L-arginine depend on a post-transcriptional and a transcriptional process, respectively. CONCLUSION: We conclude that both reduced arginine availability per se and the presence of polycationic proteins may promote airway inflammation by enhanced pro-inflammatory mediator production in airway epithelial cells, but due to distinct mechanisms.

Cytoskeletal architecture differentially controls post-transcriptional processing of IL-6 and IL-8 mRNA in airway epithelial-like cells.

Airway epithelial cells are critically dependent on an intact cytoskeleton for innate defense functions. There are various pathophysiological conditions that affect the cytoskeletal architecture. We studied the effect of cytoskeletal distortion in polarized airway epithelial-like NCI-H292 cells on inflammatory gene expression, exemplified by interleukin(IL)-6 and IL-8. Disruption of microtubule structure with vinblastin and of actin with cytochalasin D did not affect TNF-alpha-induced IL-6 and IL-8 gene transcription but stabilized IL-8 and IL-6 mRNA. In line with previous studies, IL-8 mRNA stabilization was paralleled by hyperresponsive IL-8 production, but surprisingly, IL-6 production was reduced despite IL-6 mRNA stabilization. Polysome profiling revealed that, in cells with a disrupted cytoskeleton, translational efficiency of IL-6 mRNA was reduced, whereas that of IL-8 mRNA remained unaffected. Our findings indicate that distortion of the cytoskeleton in airway epithelial cells differentially affects both degradation and translation of IL-6 and IL-8 mRNA, modifying inflammatory gene expression and thus their innate defense function.

E1A expression dysregulates IL-8 production and suppresses IL-6 production by lung epithelial cells.

BACKGROUND: The adenoviral protein E1A has been proposed to play a role in the pathophysiology of COPD, in particular by increasing IL-8 gene transcription of lung epithelial cells in response to cigarette smoke-constituents such as LPS. As IL-8 production is also under tight post-transcriptional control, we planned to study whether E1A affected IL-8 production post-transcriptionally. The production of IL-6 by E1A-positive cells had not been addressed and was studied in parallel. Based on our previous work into the regulation of IL-8 and IL-6 production in airway epithelial cells, we used the lung epithelial-like cell line NCI-H292 to generate stable transfectants expressing either E1A and/or E1B, which is known to frequently co-integrate with E1A. We analyzed IL-8 and IL-6 production and the underlying regulatory processes in response to LPS and TNF-alpha. METHODS: Stable transfectants were generated and characterized with immunohistochemistry, western blot and flow cytometry. IL-8 and IL-6 protein production was measured by ELISA. Levels of IL-8 and IL-6 mRNA were measured using specific radiolabeled probes. EMSA was used to assess transcriptional activation of relevant transcription factors. Post-transcriptional regulation of mRNA half-life was measured by Actinomycin D chase experiments. RESULTS: Most of the sixteen E1A-expressing transfectants showed suppression of IL-6 production, indicative of biologically active E1A. Significant but no uniform effects on IL-8 production, nor on transcriptional and post-transcriptional regulation of IL-8 production, were observed in the panel of E1A-expressing transfectants. E1B expression exerted similar effects as E1A on IL-8 production. CONCLUSION: Our results indicate that integration of adenoviral DNA and expression of E1A and E1B can either increase or decrease IL-8 production. Furthermore, we conclude that expression of E1A suppresses IL-6 production. These findings question the unique role of E1A protein in the pathophysiology of COPD, but do not exclude a role for adenoviral E1A/E1B DNA in modulating inflammatory responses nor in the pathogenesis of COPD.

Interleukin-17 induces hyperresponsive interleukin-8 and interleukin-6 production to tumor necrosis factor-alpha in structural lung cells.

Lung epithelial cells contribute to local inflammation by the production of pro-inflammatory mediators like interleukin (IL)-8 and IL-6. Although their production depends on gene transcription, previous studies showed that post-transcriptional mechanisms modulate IL-8 and IL-6 production. Human lung epithelial cells turn from normoresponsive into hyperresponsive IL-8- and IL-6-producing cells when their IL-8 and IL-6 mRNA degradation is reduced. We hypothesized that IL-17, a mediator predominantly released by memory T cells and present in airways of individuals with asthma, would modulate rather than induce IL-8 and IL-6 production by both human lung epithelial cells and fibroblasts. We show here for both cell types that IL-17 was a weak stimulus of IL-8 and IL-6 production, but markedly enhanced IL-8 and IL-6 responses to another stimulus, such as tumor necrosis factor-alpha. This modulatory effect of IL-17 was paralleled by a reduced IL-8 and IL-6 mRNA degradation, with no effect on IL-8 and IL-6 gene transcription. In conclusion, IL-17 particularly affects post-transcriptional regulation of IL-8 and IL-6 expression leading to enhanced IL-8 and IL-6 responses to secondary stimuli, and is only a weak proinflammatory stimulus by itself. This poses the interesting concept that by releasing IL-17 from memory T cells, the adaptive immune system instructs lung structural cells as part of the innate immune system to respond more vigorously.

Exaggerated IL-8 and IL-6 responses to TNF-alpha by parainfluenza virus type 4-infected NCI-H292 cells.

Respiratory viruses induce and potentiate airway inflammation, which is related to the induction of proinflammatory mediators such as interleukin (IL)-8 and IL-6. Here we report on mechanisms implicated in IL-8 and IL-6 production by airway epithelium-like NCI-H292 cells exposed to parainfluenza virus type 4a (PIV-4). PIV-4 readily infected NCI-H292 cells as reflected by intracellular PIV-4 antigen expression. PIV-4 infection triggered a biphasic IL-8 and IL-6 mRNA response. Transient transfection with truncated and mutated promoter constructs identified NF-kappaB and activator protein (AP)-1, and CCAAT-enhancer binding protein (C/EBP) as the relevant transcription factors for PIV-4-induced IL-8 and IL-6 gene transcription, respectively. An increase of DNA-binding activities for NF-kappaB and C/EBP paralleled the induction of the first and second IL-8 and IL-6 mRNA peaks, whereas the onset of AP-1 paralleled the first IL-8 mRNA peak only. The second mRNA peak, apparently dependent on viral replication, coincided also with a marked reduction of IL-8 and IL-6 mRNA degradation. Importantly, cells at the time of the reduced mRNA degradation displayed an exaggerated IL-8 and IL-6 protein production to a secondary stimulus, as exemplified by steeper dose-response curves to TNF-alpha. Thus PIV-4 infection enhances epithelial IL-8 and IL-6 production by transcriptional and posttranscriptional mechanisms. The previously unrecognized phase of reduced IL-8 and IL-6 mRNA degradation and the concurrent amplified epithelial IL-8 and IL-6 responses may play an important role in virus-induced potentiation of airway inflammation.

IFN-gamma amplifies IL-6 and IL-8 responses by airway epithelial-like cells via indoleamine 2,3-dioxygenase.

Respiratory viral infections increase inflammatory responses to concurrent or secondary bacterial challenges, thereby worsening disease outcome. This potentiation of inflammation is explained at least in part by IFN-gamma promoting increased sensitivity to TNF-alpha and LPS. We sought to determine whether and, if so, how IFN-gamma can modulate proinflammatory responses to TNF-alpha and LPS by epithelial cells, which are key effector cells in the airways. Preincubation of airway epithelial-like NCI-H292 cells with IFN-gamma resulted in a hyperresponsive IL-6 and IL-8 production to TNF-alpha and LPS. The underlying mechanism involved the induction of indoleamine 2,3-dioxygenase, which catabolized the essential amino acid, tryptophan. Depletion of tryptophan led to stabilization of IL-6 and IL-8 mRNA and increased IL-6 and IL-8 responses, whereas supplementing tryptophan largely restored these changes. This novel mechanism may be implicated in enhanced inflammatory responses to bacterial challenges following viral infection.