Human peritoneal mesothelial cells synthesize interleukin-8. Synergistic induction by interleukin-1 beta and tumor necrosis factor-alpha.

The present study demonstrates the synthesis and secretion of the neutrophil-activating peptide/interleukin-8 (IL-8) by cultured human peritoneal mesothelial cells (HPMC) and examines the regulation of its production by other cytokines. Unstimulated HPMC under growth-arrested conditions released IL-8 in a constitutive and time-dependent manner. Stimulation of HPMC with IL-1 beta or TNF-alpha resulted in a time- and dose-dependent IL-8 generation; after 24 hours the levels induced by IL-1 beta and TNF-alpha (both at 1000 pg/ml) were (mean +/- SEM, n = 5) 101 +/- 26.6 (z = 2.023; P < 0.01) and 35 +/- 8.09 (z = 2.023; P < 0.01) respectively. This release was inhibited following coincubation with the relevant anti-cytokine antibody or preincubation with either cycloheximide or actinomycin D. Treatment of HPMC with IL-1 beta or TNF-alpha resulted in increased levels of IL-8-specific mRNA. Stimulation of HPMC with combinations of IL-1 beta and TNF-alpha resulted in a synergistic increase in IL-8 release. This effect was significant at combined doses of IL-1 beta (50 pg/ml) and TNF-alpha (500 pg/ml) and above, when the release of IL-8 was 88 +/- 27% above the additive IL-8 release values (z = 2.201; P < 0.01). Western blot analysis using specific anti-IL-8 antibody demonstrated the presence of two major immunoreactive bands between 9 and 10 kd, in HPMC culture supernatants. These data demonstrate that HPMC synthesize IL-8 and that its release can be regulated as a result of induction of mRNA expression and de novo protein synthesis by other cytokines.     

Cellular localization of interleukin-8 and its inducer, tumor necrosis factor-alpha in psoriasis.

The importance of immunologic mechanisms in psoriasis has been deduced from the ability of immunosuppressive therapies to ameliorate this common and chronic skin disease. Certainly the histology of psoriatic lesions suggests a dialogue between the hyperplastic keratinocytes and infiltrating T lymphocytes and macrophages. To begin dissecting the cytokine network involved in the pathophysiology of psoriasis, the location, in both epidermal and dermal compartments, of tumor necrosis factor-alpha, interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha at the protein and/or mRNA levels were identified. Tumor necrosis factor-alpha was selected as a potentially key regulatory cytokine, first because it induces cultured keratinocyte interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha production, and second because intercellular adhesion molecule-1 expression by keratinocytes in psoriatic epidermis had been identified previously. Using immunohistochemical localization, tumor necrosis factor-alpha was identified in 12 psoriatic lesions as intense and diffuse expression by dermal dendrocytes (macrophages) in the papillary dermis (without significant staining of endothelial cells, mast cells, or dermal Langerhans cells), and focally by keratinocytes and intraepidermal Langerhans cells. Functional interaction between the dermal dendrocytes and keratinocytes was suggested by the presence of interleukin-8 expression of suprabasal keratinocytes immediately above the tumor necrosis factor-alpha-positive dermal dendrocytes. Interleukin-8 mRNA and transforming growth factor-alpha mRNA were detectable in the epidermal roof of psoriatic lesions, but neither was detectable at the protein or mRNA levels in any normal skin specimens. Treatment of cultured human keratinocytes with phorbol ester (which experimentally produces psoriasiform changes on mouse skin) or tumor necrosis factor-alpha also increased interleukin-8 and transforming growth factor-alpha mRNAs. Further elucidation of the cellular and molecular basis for the genesis and evolution of psoriasis will provide the framework for a better evaluation of the cause and treatment of this skin disease.     

Production of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by a rat corneal epithelial cell line

Production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by rat corneal epithelial cells in response to lipopolysaccharide and phorbol-12-myristate-13-acetate (PMA) was tested. Supernatants from rat corneal epithelial cells treated with lipopolysaccharide and PMA were collected after 6, 24 and 48 h and tested with enzyme-linked immunosorbent assay for IL-1 beta, IL-6 and TNF-alpha. The activity of TNF-alpha was additionally confirmed with bioassay on L929 cells. It was found that control groups did not produce significant levels of either cytokine. However, after stimulation with lipopolysaccharide, cells produced mainly IL-6, whereas after PMA they produced mainly TNF-alpha. IL-6 levels 24 and 48 h after PMA stimulation were also elevated, which could have been caused by the presence of TNF-alpha. Production of IL-1 beta in all groups was very low and remained within the test sensitivity range. These results show that the rat corneal epithelial cell line produces inflammatory cytokines in response to proinflammatory mediators. For this reason, it could be used for measuring the effects of irritants on the cornea.     

Human alveolar macrophage gene expression of interleukin-8 by tumor necrosis factor-alpha, lipopolysaccharide, and interleukin-1 beta

The alveolar macrophage (AMO) in its pivotal position for pulmonary host defense may play a prominent role in the orchestration of polymorphonuclear leukocyte (PMN) diapedesis. We demonstrate that the human AMO may participate in these inflammatory events through the production of a novel neutrophil chemotactic factor, interleukin-8 (IL-8). The induction of AMO-derived IL-8 by tumor necrosis factor (TNF), lipopolysaccharide (LPS), and interleukin-1 (IL-1 beta) was shown to be both dose and time dependent. Maximal IL-8 gene expression, as assessed by Northern blot analyses, was achieved with 20 ng/ml and 1 microgram/ml, respectively, for each of the cytokines and LPS. A kinetic study of TNF-, IL-1 beta-, and LPS-treated AMOs showed significant steady-state IL-8 mRNA accumulation post-stimulation at 1 h, peaking by 8 h, with a decline over the next 16 h. Immunohistochemical staining using rabbit anti-human IL-8 antibody demonstrated significant immunolocalization of cell-associated IL-8 antigen at 4 h, with persistence over the next 20 h. Chemotactic bioactivity peaked by 8 h, with continued production over the next 16 h. Chemotactic bioactivity from AMO-conditioned media was inhibited by IL-8 antiserum by 2, 31, 44, and 47%, respectively, for unstimulated control, LPS-, IL-1 beta-, and TNF-treated cells. Preimmune serum had no effect on chemotactic activity. These data support the central role of the AMO in the elicitation of PMNs into the lung via the production of IL-8.     

Autocrine regulation of interleukin-8 by interleukin-1alpha in respiratory syncytial virus-infected pulmonary epithelial cells in vitro.

Respiratory epithelial cells infected with respiratory syncytial virus (RSV) produce interleukin-8 (IL-8); however, the mechanisms of RSV-induced regulation of IL-8 are poorly understood. In the present study, the regulation of IL-8 by RSV was evaluated using pulmonary type II-like epithelials (A549). Live purified RSV (pRSV) induced a significant increase in IL-8 after 8 hr of exposure, while conditioned supernatants from pRSV-infected A549 cells (cRSV) induced IL-8 production in fresh A549 cultures within 4 hr of infection. Furthermore, cRSV that had been rendered non-infectious by ultraviolet-irradiation (UV-cRSV) or ribavirin treatment also induced an increased production of IL-8 in fresh A549 cells, suggesting that RSV induced the synthesis of a soluble mediator(s) which in turn enhanced the synthesis of IL-8. We have previously shown that RSV-infected A549 cells produce IL-1alpha, IL-1-beta and tumour necrosis factor-alpha (TNF-alpha), which by themselves are known to induce the synthesis of IL-8. Preincubation of UV-cRSV or simultaneous incubation of pRSV with recombinant IL-1 receptor antagonist almost completely blocked (95-98%) the production of IL-8 by A549 cells. Furthermore, incubation with neutralizing antibodies against IL-1alpha, IL-1beta and TNF-alpha showed that IL-1alpha was the predominant soluble mediator that enhanced the mRNA expression and synthesis of IL-8. IL-1beta and TNF-alpha induced the synthesis of IL-8 at 24 hr, but partially inhibited the synthesis at 48 hr. In summary, these experiments provide direct evidence for an autocrine mechanism of enhanced IL-8 production in RSV-infected epithelial cells that is primarily mediated by IL-1alpha. In clinical settings, inhibitors of IL-1alpha may be useful in suppressing inflammation due to IL-1alpha as well as IL-8.     

Tumour necrosis factor-alpha, interleukin-6 and interleukin-8 do not promote adhesion of human endometrial epithelial cells to mesothelial cells in a quantitative in vitro model

BACKGROUND: A key factor in the pathogenesis of endometriosis is the endometrial-peritoneal adhesion. To study the pathogenesis of endometriosis, a quantitative in vitro assay (QIVA) was developed to measure in vitro adhesion between human endometrial epithelial cells and mesothelial cells using commercially available cell lines. Using the QIVA, the hypothesis was tested that tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and interleukin-8 (IL-8) promote adhesion of endometrial epithelial cells to mesothelial cells. METHODS: Mesothelial cells were pre-treated with TNF-alpha, IL-6 or IL-8 in various concentrations (ranging from 0 to 1000 IU/ml) for 24 h. Confluent endometrial epithelial cells were labelled with [35S]methionine, added to the confluent mesothelial cells and incubated for 1 h. After incubation, non-adhering cells were removed and adherent cells were solubilized and their [35S]methionine radioactivity was counted to quantify the adherence of endometrial epithelial cells to mesothelial cells. RESULTS: The in vitro adhesion of human endometrial epithelial cells to human mesothelial cells was inhibited in a dose-dependent manner by TNF-alpha (P=0.0007), IL-6 (P<0.0001) and IL-8 (P=0.0004). CONCLUSIONS: Using a quantitative in vitro adhesion assay, we were unable to confirm our hypothesis that TNF-alpha, IL-6 and IL-8 promote the in vitro adhesion between endometrial epithelial cells and mesothelial cells.     

Indirect and selective down-regulation of serum tumor necrosis factor-alpha release by interleukin-1 beta.

A selective inhibition of LPS-induced tumor necrosis factor-alpha (TNF) response in mice was caused by an injection of recombinant human interleukin-1 (IL-1). The decrease in serum TNF level reached 70 to 80 percent of the controls receiving LPS alone when IL-1 was given simultaneously or prior to the challenge. At the same time serum IL-6 release was more elevated. Ex vivo assays have shown that macrophages from IL-1 treated animals did not respond to LPS when stimulated immediately after harvesting but recovered their normal responsiveness after being cultured for 2 hours and then washed. In vitro with or without addition of IL-1, mouse elicited macrophages responded equally to LPS in releasing TNF. In the absence of a direct and lasting effect on TNF-producing cells, the host reaction responsible for the inhibitory effect of IL-1 could be related to the overproduction of corticosterone that occurred after IL-1 injection, since it was not observed in adrenalectomized animals. Indeed the blockade of corticoid secretion by indomethacin prevented the inhibition of TNF production induced by IL-1 administration before LPS challenge. TNF administration did not result in elevation of corticosterone level and in contrast to IL-1 enhanced the TNF response to LPS injection. In vitro and ex vivo assays have shown this enhanced response to LPS was linked to a direct and prolonged effect of TNF on TNF-producing cells. Muramyl dipeptide (MDP) which was used as a known priming agent for enhanced cytokine release had a similar effect on TNF-producing cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of cadherin and catenins expression by tumor necrosis factor-alpha and dexamethasone in human bronchial epithelial cells

Asthma is an inflammatory disease, and the epithelial mesenchymal unit appears to be of importance in regulating the disease mechanisms. Cell-cell adhesion plays an important role in tissue morphogenesis and homeostasis and is commonly mediated by cadherins, a family of Ca(2+)-dependent transmembrane adhesion receptors. The cadherin family is involved in control of the cellular architecture. Proinflammatory cytokines such as tumor necrosis factor (TNF)-alpha are involved in asthma and may interfere with epithelial integrity. In the present study, we investigated the role of TNF-alpha and dexamethasone on the expression of E-cadherin, beta-catenin, and gamma-catenin. We used two bronchial epithelial cell models: primary small airway epithelial cell cultures and primary culture obtained from human bronchial tubes. After 48 h of TNF-alpha stimulation with or without dexamethasone expression of E-cadherin, beta-catenin and gamma-catenin were analyzed using Western blot analysis and immunofluorescence. This study showed a decrease in the expression of adhesion molecules in both epithelial cell cultures after stimulation. Dexamethasone and anti-TNF-alpha inhibited this effect. In unstimulated cells, E-cadherin and beta- and gamma-catenin expression was membranous, expressed only on the lateral cell wall with minimal cytoplasmic expression. Immunoreactivity was cytoplasmic in stimulated cells. We demonstrated, using Western blot analysis and immunofluorescence, that proinflammatory cytokines could be responsible for structural damage to the epithelium and that this process was potentially reversed by steroids.     

Requirements for tumor necrosis factor-alpha and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury.

Ischemia in rat hind limbs followed by reperfusion results in local as well as remote organ (lung) injury characterized by increased vascular permeability (125I-labeled bovine serum albumin leakage) and hemorrhage (51Cr-labeled rat erythrocytes extravasation) in skeletal muscle and lung, together with an associated increased tissue content of myeloperoxidase, reflecting neutrophil accumulation. Within 60 minutes of reperfusion following ischemia, tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 plasma levels increased significantly, reaching maximum levels after 2 hours of reperfusion. Polyclonal antibodies to TNF-alpha and IL-1 provided significant protection against vascular injury in both muscle and lung. These results were confirmed by the use of soluble TNF-alpha receptor and IL-1 receptor antagonist. In rat lungs following ischemia and reperfusion, there was immunohistochemical evidence of E-selectin expression in the lung vasculature; this expression was blocked by treatment of animals with anti-TNF-alpha. These data indicate that both local (hind limb) and remote (lung) organ injury after ischemia/reperfusion requires participation of TNF-alpha and IL-1. The cytokines may, in part, be involved in the up-regulation of endothelial adhesion molecules.     

Detection of in vivo production of tumour necrosis factor-alpha by human thyroid epithelial cells.

We have established previously that human thyroid epithelial cells (TEC) from patients with autoimmune thyroiditis are able to synthesize cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6). This paper examines TEC in sections from autoimmune thyroiditis for the in vivo production of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using the combined techniques of in situ hybridization and immunohistochemistry. Thyroid tissue from patients with Graves' disease, Hashimoto's disease and non-toxic goitre was examined and both mRNA and the protein of TNF-alpha were detected in TEC on frozen sections. Representative figures of only Graves' samples are illustrated in this paper. In contrast, using the same methods, IFN-gamma was detected only in the infiltrating cells and not in TEC of thyroid tissue from the patients.     

Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1 beta and tumour necrosis factor-alpha.

We have tested the hypothesis that the bronchial epithelium has the capacity to generate and release cytokines that could contribute to inflammatory events associated with inflammatory lung diseases. Messenger RNA (mRNA) for interleukin-6 (IL-6), IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) was identified in human bronchial epithelial cell primary cultures, characterized on the basis of staining for cytokeratin, using both in situ hybridization and Northern blotting. Using in situ hybridization we have shown that the majority of the cells expressed mRNA for IL-6 and IL-8, whereas fewer than 20% of cells expressed message for GM-CSF. The numbers of cells expressing message were increased by culture with tumour necrosis factor-alpha (TNF-alpha) (20 ng/ml, 24 hr). These observations were substantiated by Northern blotting, which showed that both TNF-alpha and IL-1 beta were able to induce a dose-dependent increase in IL-8-specific mRNA. Immunoreactive IL-6 and GM-CSF were detected and quantified in the culture supernatants by ELISA, and IL-8 by radioimmunoassay. The levels of immunoreactivity were increased by incubation of epithelial cells with either IL-1 beta or TNF-alpha for 24 hr. A transformed tracheal epithelial cell line (9HTEo-) expressed mRNA for IL-6, IL-8 and GM-CSF but, whereas levels of immunoreactive IL-6 in culture supernatants were comparable with those in primary cell cultures, levels of IL-8 were low and GM-CSF trivial. These observations indicate that the bronchial epithelium has the potential to be a major source of IL-8 and a number of other cytokines, and that production can be amplified substantially by IL-1 beta and TNF-alpha. The bronchial epithelium is ideally situated to modulate inflammatory and immunological events in and around the airways, and these observations suggest that it could contribute to promote and sustain inflammatory and immunological processes in inflammatory lung diseases such asthma.     

Cytotoxicity of tumor necrosis factor for thyroid epithelial cells and its regulation by interferon-gamma

The FRTL-5 line of differentiated rat thyroid epithelial cells was shown to be sensitive to the cytotoxic action of recombinant tumor necrosis factor. The sensitivity of the cells varied with the conditions of culture. It was markedly increased by preincubation with recombinant interferon-gamma, and the magnitude of this enhancement was affected by the presence of thyroid-stimulating hormone. The increased susceptibility of the cells to the cytotoxicity of tumor necrosis factor was clear as early as 2-4 h after the addition of interferon-gamma and greatly preceded the induced expression of major histocompatibility complex class II antigen. Since cells secreting interferon-gamma and tumor necrosis factors may co-exist in inflammatory infiltrates, our observations suggest that these factors may be early mediators of cell destruction in autoimmune thyroid disease.     

Tumor necrosis factor-alpha is expressed by glomerular visceral epithelial cells in human membranous nephropathy.

The role of tumor necrosis factor alpha (TNF-alpha) was examined in biopsy-proven glomerulonephritis by immunohistochemistry, in situ hybridization, immunogold electron microscopy, immunoassay in serum and urine, and urinary immunoblot. Striking glomerular capillary wall and visceral glomerular epithelial cell TNF-alpha protein staining was observed in all cases of membranous nephropathy and membranous lupus nephropathy. Staining was less frequently observed in crescentic glomerulonephritis and in isolated cases of other histological subtypes of glomerulonephritis, usually in association with glomerular macrophages. By immunogold electron microscopy TNF-alpha was localized in membranous nephropathy within the visceral glomerular epithelial cells, and also in the glomerular basement membrane, especially in relation to immune deposits. In situ hybridization localized TNF-alpha mRNA exclusively to glomerular epithelial cells in all biopsies with membranous morphology but not in other histological subtypes. Concentrations of TNF-alpha were significantly increased compared with normal controls in the urine of patients with membranous nephropathy and with crescentic glomerulonephritis. The expression of TNF-alpha by glomerular epithelial cells exclusively and universally in biopsies showing a membranous morphology strongly suggests this cytokine has a role in the pathogenesis of membranous nephropathy.