Epithelial overexpression of interleukin-32alpha in inflammatory bowel disease

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by induction of nuclear factor (NF)-kappaB activation. We studied IL-32alpha expression in the inflamed mucosa of inflammatory bowel disease (IBD). We also investigated mechanisms regulating IL-32alpha expression. Tissue samples were obtained endoscopically or surgically from patients with ulcerative colitis (UC) (n = 10), Crohn's disease (CD) (n = 10), ischaemic colitis (n = 4) and normal colorectal tissues (n = 10). IL-32alpha expression was evaluated by standard immunohistochemical procedure. IL-32 mRNA expression was analysed by Northern blot. IL-32alpha was expressed weakly by colonic epithelial cells from normal individuals and subjects with ischaemic colitis. In the inflamed mucosa of IBD patients, epithelial IL-32alpha expression was increased markedly. In UC and CD patients, IL-32alpha expression was enhanced in affected mucosa compared to non-affected mucosa. In intestinal epithelial cell lines, expression of IL-32alpha mRNA and protein was enhanced by IL-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. A combination of TNF-alpha plus IFN-gamma exerted synergistic effects. IL-32alpha induction by IL-1beta and/or TNF-alpha was mediated by NF-kappaB activation. Epithelial IL-32alpha expression was increased in IBD patients, and in CD patients in particular. IL-32alpha might be involved in the pathophysiology of IBD as a proinflammatory cytokine and a mediator of innate immune response.     

Enhanced chemokine response in experimental acute Escherichia coli pyelonephritis in IL-1beta-deficient mice

The aim of the present study was to investigate the effects of IL-1beta and Escherichia coli on the expression and secretion of MIP-2, the mouse equivalent to human IL-8, MCP-1 and RANTES in the kidneys of mice with acute pyelonephritis. Female Bki NMRI, as well as IL-1beta deficient mice and their wild-type littermates, were transurethrally infected with either E. coli CFT 073 or injected with NaCl 0.9% (w/v) and thereafter obstructed for 6 h. The Bki NMRI mice were killed at 0, 24, 48 h and 6 days and the IL-1beta-deficient mice at 48 h. Chemokine mRNA and protein levels peaked at 24 h for the tested chemokines with the mRNA expression localized in the tubular epithelial cells and for MIP-2 also in neutrophils. Obstruction per se, also induced a chemokine expression similar to E. coli infection although at a lower level. Interestingly, MIP-2 levels were higher in the IL-1beta deficient mice as compared with the wild-type littermates. Likewise, the inflammatory changes were more frequent and, when present, more widespread in the IL-1beta-deficient mice than in the wild-type mice. Stimulation of a human renal tubular epithelial cell line (HREC), A498 and of primary human mesangial cells (HMC) with the same bacterial antigen depicted gene expression of the same chemokines. A rapid release of IL-8 and MCP-1 was observed from both cell types. RANTES response was delayed both in the HREC and the HMC. We conclude that acute E. coli pyelonephritis induces a MIP-2/IL-8, MCP-1 and RANTES expression and secretion localized primarily to the epithelial cells and that this production is confirmed after in vitro stimulation with the same bacterial antigen of human epithelial and mesangial cells. Blockade of induction of chemokine response may thus be an attractive target for possible therapeutic intervention.     

Nasal epithelial cells as surrogates for bronchial epithelial cells in airway inflammation studies

The nose is an attractive source of airway epithelial cells, particularly in populations in which bronchoscopy may not be possible. However, substituting nasal cells for bronchial epithelial cells in the study of airway inflammation depends upon comparability of responses, and evidence for this is lacking. Our objective was to determine whether nasal epithelial cell inflammatory mediator release and receptor expression reflect those of bronchial epithelial cells. Paired cultures of undifferentiated nasal and bronchial epithelial cells were obtained from brushings from 35 subjects, including 5 children. Cells were subject to morphologic and immunocytochemical assessment. Mediator release from resting and cytokine-stimulated cell monolayers was determined, as was cell surface receptor expression. Nasal and bronchial cells had identical epithelial morphology and uniform expression of cytokeratin 19. There were no differences in constitutive expression of CD44, intercellular adhesion molecule-1, alphavbeta3, and alphavbeta5. Despite significantly higher constitutive release of IL-8, IL-6, RANTES (regulated on activation, normal T cell expressed and secreted), and matrix metalloproteinase (MMP)-9 from nasal compared with bronchial cells, the increments in release of all studied mediators in response to stimulation with IL-1beta and TNF-alpha were similar, and there were significant positive correlations between nasal and bronchial cell secretion of IL-6, RANTES, vascular endothelial growth factor, monocyte chemoattractant protein-1, MMP-9, and tissue inhibitor of metalloproteinase-1. Despite differences in absolute mediator levels, the responses of nasal and bronchial epithelial cells to cytokine stimulation were similar, expression of relevant surface receptors was comparable, and there were significant correlations between nasal and bronchial cell mediator release. Therefore, nasal epithelial cultures constitute an accessible surrogate for studying lower airway inflammation.     

C-X-C and C-C chemokine expression and secretion by the human colonic epithelial cell line, HT-29: differential effect of T lymphocyte-derived cytokines

Differential chemokine production by colonic epithelial cells is thought to contribute to the characteristic increased infiltration of selected population of leukocytes cells in inflammatory bowel disease. We have previously demonstrated that IL-13 enhances IL-1alpha-induced IL-8 secretion by the colonic epithelial cell line HT-29. We have now explored the C-C chemokine expression and modulation in this system. The combination of TNF-alpha and IFN-gamma was the minimal stimulation required for regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemoattractant protein (MCP-1) mRNA expression and secretion by HT-29 cells. The same stimulation induced a stronger IL-8 mRNA expression and secretion. Pretreatment with IL-13 or IL-4, reduced significantly the RANTES, and MCP-1, but not IL-8 mRNA expression and secretion. In contrast, IL-10 had no effect on either MCP-1, or RANTES, or IL-8 generation. Pretreatment of HT-29 cells with wortmannin suggested that the IL-13-induced inhibition of C-C chemokine expression is via activation of a wortmannin-sensitive phosphatidylinositol 3-kinase. These data demonstrate that colonic epithelial cell chemokine production can be differentially regulated by T cell-derived cytokines and suggest an interplay between epithelial cells and T lymphocytes potentially important in the intestinal inflammation.     

Enhancing effect of cholera toxin on interleukin-6 secretion by IEC-6 intestinal epithelial cells: mode of action and augmenting effect of inflammatory cytokines.

Oral administration of cholera toxin (CT) induces a strong mucosal immune response to CT as well as having a potent adjuvant effect. Since one of the first cell types to encounter CT during cholera infection or after oral administration is the epithelial cell, we studied the effect of CT on interleukin-6 (IL-6) secretion by the rat intestinal epithelial cell line IEC-6. CT was found to rapidly enhance IL-6 secretion and IL-6 gene expression by these cells. The addition of dibutyryl cyclic AMP (cAMP) to cultures of IEC-6 cells had little effect on IL-6 secretion, yet mRNA levels were elevated, suggesting that the response may have been regulated by cAMP. Purified B subunit of CT did not significantly enhance IL-6 secretion or mRNA expression. CT and transforming growth factor beta 1 synergistically enhanced IL-6 secretion in IEC-6 cells. The addition of CT with either IL-1 beta or tumor necrosis factor alpha gave even greater synergistic enhancement of IL-6 secretion, and dibutyryl cAMP could mimic CT's synergy with IL-1 beta. These results indicate that the intestinal epithelial cell is capable of secreting high levels of IL-6 after encountering CT, especially in the presence of inflammatory cytokines. This high level of IL-6 secretion could be a very important component of the mucosal immune response to CT and may also account for a portion of the adjuvant effect of CT.     

Colonic epithelial cell lines as a source of interleukin-8: stimulation by inflammatory cytokines and bacterial lipopolysaccharide.

Cytokines produced by intestinal epithelial cells may function as signals to neighbouring immune and inflammatory cells. We investigated production of the neutrophil and T-lymphocyte chemotactic cytokine interleukin-8 (IL-8) by intestinal epithelial cells using four colonic adenocarcinoma cell lines, T84, CaCo-2, HT29 and SW620, as a model system. These cell lines secreted substantial amounts of IL-8 if stimulated with IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma), except CaCo-2 cells, which responded only to IL-1 beta. Bacterial lipopolysaccharide (LPS) was also an efficient stimulus of IL-8 release in SW620 and HT29 cells, whereas T84 and CaCo-2 cells were completely unresponsive to LPS, IL-8 secretion was greater at 4 hr after stimulation and was accompanied by induction of IL-8 messenger RNA. In T84 cells IFN-gamma and epidermal growth factor (EGF) stimulated IL-8 secretion synergistically with TNF-alpha, whereas in SW620 cells this synergism occurred only between IFN-gamma and TNF-alpha. IL-4, IL-10 and transforming growth factor-beta (TGF-beta), which can down-regulate IL-8 production in macrophages, had no effect on IL-8 generation by our cell lines. Adenocarcinoma cell culture supernatants also induced rapid transients of intracellular calcium in neutrophils. Depending on cell line and stimulus, supernatant bioactivity was completely or partially abrogated by neutralizing antibodies to IL-8, indicating that the cell lines investigated also generate other neutrophil-activating factors. IL-8 and possibly other chemokines generated by colonic adenocarcinomas may help to attract tumour-infiltrating leucocytes. Possibly, normal intestinal epithelial cells also have the potential to secrete this potent chemoattractant and thus might contribute to inflammatory responses of the intestinal mucosa, for example in inflammatory bowel disease.     

Cytokine-induced gene expression of interleukin-8 in human transitional cell carcinomas and renal cell carcinomas.

Chemotactic cytokines play a critical role in recruiting leukocytes to sites of tissue injury. Interleukin-8 (IL-8) is a chemotactic cytokine secreted by a variety of cells (eg, monocytes, endothelial cells, fibroblasts) during the inflammatory response. In this report, the authors demonstrate that human transitional cell carcinomas and renal cell carcinomas have the capacity to elaborate IL-8 in response to the inflammatory mediators IL-1 beta and tumor necrosis factor (TNF)-alpha. All cell lines expressed high levels of IL-8 mRNA on stimulation with either IL-1 beta or TNF-alpha, but not lipopolysaccharide; one expressed the gene constitutively. The authors selected one transitional cell carcinoma cell line (UM-UC-9) and one renal cell carcinoma cell line (UM-RC-5) for further study. Both displayed a time- and dose-dependent increase in steady-state levels of IL-8 mRNA in response to IL-1 beta and TNF-alpha. Specific mRNA was detectable by 1 hour after stimulation. Secretion of antigenic IL-8 measured by enzyme-linked immunosorbent assay into culture supernatants reflected the kinetics of mRNA expression. Because heat-inactivated TNF-alpha failed to induce synthesis of IL-8 mRNA, and cycloheximide augmented TNF-alpha-induced synthesis, IL-8 expression appears to be a stimulus-specific primary induction phenomenon. As with other inflammatory mediators whose mRNA contains a 3' AU-rich sequence (eg, IL-2, TNF-alpha), the half-life of IL-8 mRNA was short, less than 1 hour. Our data suggest that secretion of IL-8 by malignant cells may partly account for the inflammatory infiltrates associated with some malignant neoplasms.     

Induction of proinflammatory cytokines from human respiratory epithelial cells after stimulation by nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae (NTHi) causes repeated respiratory infections in patients with chronic lung diseases. These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the lungs and is dependent on the expression and secretion of proinflammatory cytokines. We hypothesize that multiple NTHi molecules, including lipooligosaccharide (LOS), mediate cellular interactions with respiratory epithelial cells, leading to the production of proinflammatory cytokines. To address this hypothesis, we exposed 9HTEo- human tracheal epithelial cells to NTHi and compared the resulting profiles of cytokine gene expression and secretion using multiprobe RNase protection assays and enzyme-linked immunosorbent assays (ELISA), respectively. Dose-response experiments demonstrated a maximum stimulation of most cytokines tested, using a ratio of 100 NTHi bacterial cells to 1 9HTEo- tracheal epithelial cell. Compared with purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold increases in epithelial cell expression of interleukin-8 (IL-8) and IL-6 genes, respectively. Similar results were seen with epithelial cell macrophage chemotactic protein 1, IL-1alpha, IL-1beta, and tumor necrosis factor alpha expression. Polymyxin B completely inhibited LOS stimulation but only partially reduced NTHi whole cell stimulation. Taken together, these results suggest that multiple bacterial molecules including LOS contribute to the NTHi stimulation of respiratory epithelial cell cytokine production. Moreover, no correlation was seen between NTHi adherence to epithelial cells mediated by hemagglutinating pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These data suggest that bacterial molecules beyond previously described NTHi cell surface adhesins and LOS play a role in the induction of proinflammatory cytokines from respiratory epithelial cells.     

Matrix metalloproteinase inhibitor regulates inflammatory cell migration by reducing ICAM-1 and VCAM-1 expression in a murine model of toluene diisocyanate-induced asthma

BACKGROUND: Matrix metalloproteinase-9 (MMP-9) has been reported to play a crucial role in the transmigration of neutrophils, lymphocytes, and eosinophils. Neutrophils, eosinophils, and lymphocytes migrate from the blood to the lungs in response to inflammatory mediators produced in the airways and are subsequently released into the circulation. This traffic is mediated by adhesion molecules. However, little is known about the migration of inflammatory cells through the endothelial and epithelial basement membranes in toluene diisocyanate (TDI)-induced asthma. OBJECTIVES: An aim of this study was to evaluate the effect of MMP inhibitors on the expression of ICAM-1 and VCAM-1 in the migration of inflammatory cells in a murine model of TDI-induced asthma. METHODS: We used a murine model to investigate TDI-induced asthma to examine the possible involvement of ICAM-1 and VCAM-1 in the pathogenesis of that disease and the effect of MMP inhibitors on the expression of ICAM-1 and VCAM-1. RESULTS: In mice, the following typical pathophysiologic features develop in the lungs: increased numbers of inflammatory cells and increased expression of MMP-9, ICAM-1, and VCAM-1 mRNA and protein. Administration of MMP inhibitors reduced the increased numbers of inflammatory cells and the increased expression of ICAM-1 and VCAM-1 mRNA expression and protein. In addition, MMP inhibitors significantly abrogated the increased expression of IL-1beta, IL-4, and TNF-alpha mRNA in lung tissues and levels of IL-1beta, IL-4, and TNF-alpha in bronchoalveolar lavage fluids after TDI inhalation. CONCLUSIONS: These results suggest that MMP inhibitors regulate inflammatory cell migration by reducing ICAM-1 and VCAM-1 expression and possibly also by suppressing IL-1beta, IL-4, and TNF-alpha expression.     

Neutrophil migration stimulates rat intestinal epithelial cell cytokine expression during helminth infection

We are interested in understanding the role of epithelial cells during inflammation, and we previously reported that rat small intestinal epithelial cells express interleukin-1beta (IL-1beta) during infection by Trichinella spiralis. We now report that the epithelium also produces the potent neutrophil chemotactic factor, macrophage inflammatory protein-2 (MIP-2), and an IL-1 antagonist: the type II IL-1 receptor. Consequently we investigated the pattern of neutrophil infiltration into the infected intestine, which closely paralleled the epithelial cytokine expression. Speculating that neutrophil infiltration may provoke epithelial cytokine expression, neutrophil migration into the infected gut was reduced by depleting circulating cells through the use of a specific antibody, or by preventing migration through the use of a function-blocking anti-CD18 monoclonal antibody. Either treatment reduced the number of neutrophils recoverable from the small intestinal epithelium and was paralleled by reduced mRNA levels for epithelial cytokines. These results demonstrate that neutrophil infiltration of the small intestinal epithelium contributes to the stimulation of epithelial cell cytokines.     

A synergistic relationship between TNF-alpha, IL-1 beta, and TGF-beta 1 on IL-6 secretion by the IEC-6 intestinal epithelial cell line.

Intestinal epithelial cells are known to secrete a variety of cytokines and may play a role in the immune response at the intestinal mucosa. However, the regulatory mechanisms that govern the secretion of these cytokines are largely unknown. In this report, we have focused on the cytokine interactions that regulate interleukin (IL)-6 secretion by the non-transformed rat small intestinal epithelial cell line IEC-6. Tumour necrosis factor-alpha (TNF-alpha) was found to enhance both IL-6 mRNA expression and protein secretion by the IEC-6 cells. Furthermore, TNF-alpha acted in synergy with either transforming growth factor-beta 1 (TGF-beta 1) or IL-1 beta to greatly enhance IEC-6 cell IL-6 secretion. Although the IEC-6 cells are known to produce TGF-beta, autocrine-secreted TGF-beta was found to have no effect on the elevated IL-6 secretion induced by both TNF-alpha plus IL-1 beta. However, the addition of activated TGF-beta 1 to IEC-6 cultures stimulated with both TNF-alpha and IL-1 beta resulted in greatly elevated levels of IL-6 secretion. Therefore, activated TGF-beta 1 can augment IL-6 secretion stimulated by TNF-alpha and IL-1 beta, either alone or in combination, suggesting that intestinal epithelial cell IL-6 secretion may be under the control of a cytokine network at the intestinal mucosa.     

In Situ Expression of Interleukin-10 in Noninflamed Human Gut and in Inflammatory Bowel Disease

A dysregulated secretion of contra-inflammatory cytokines such as interleukin-10 (IL-10) could play a role in the pathogenesis of inflammatory bowel disease (IBD). We have investigated the expression of IL-10 in gut tissues from patients with Crohn’s disease (CD), ulcerative colitis (UC) and controls by mRNA in situ hybridization and immunohistochemistry. Intestinal epithelial cells were found to express IL-10 mRNA and IL-10 protein in all of the tissues investigated without any major differences in the expression patterns. However, compared with noninflamed gut, significantly increased numbers of mononuclear cells (MNCs) producing IL-10 were present in inflamed gut, both in CD and UC. This cytokine was expressed most prominently by inflammatory infiltrates enriched in macrophages, although T cells seem to contribute to its production as well. Elevated IL-10 expression in IBD was mainly detected in the submucosa, whereas IL-10 production by lamina propria cells remained comparably low. In contrast, the expression of IL-1β mRNA was preferentially increased in the lamina propria. Our data argue against a general deficiency in IL-10 production in IBD. The results suggest rather that the local production of IL-10 by mucosal MNCs in IBD is insufficient to down-regulate pro-inflammatory cytokines such as IL-1β in the lamina propria compartment.     

Prolonged expression of lipopolysaccharide (LPS)-induced inflammatory genes in whole blood requires continual exposure to LPS.

Blood-borne lipopolysaccharide (LPS) is thought to be a major inducer of sepsis; however, it remains controversial whether an ongoing exposure to LPS is required to maintain the underlying systemic inflammatory response. To address this question, we have studied the expression of tumor necrosis factor alpha (TNF-alpha), interleukin 1-beta (IL-1 beta), and the procoagulant protein tissue factor induced by LPS ex vivo in whole human blood. The addition of a 1-ng/ml bolus of LPS to blood rapidly induced mRNA expression of all three genes. The mRNA levels peaked after 1 to 2 h, depending on the gene, and then declined to baseline after approximately 5 h. The decline in mRNA expression was not caused by a loss of responsiveness of the blood cells to LPS but rather correlated with the neutralization of LPS inflammatory activity by plasma components. Furthermore, administering a 1-ng/ml dose of LPS in six hourly aliquots of 167 pg/ml greatly prolonged the expression of mRNAs and induced a much greater release of TNF-alpha and IL-1 beta protein than did a single bolus. Dosing by repeated additions was more effective than a single bolus in inducing secretion of TNF-alpha and IL-1 beta at LPS levels of < or = 10 ng/ml, which corresponded to the LPS neutralization capacity of plasma. Finally, both mRNA expression and protein secretion induced by repeated administration of LPS were rapidly reversed by the addition of the LPS-neutralizing protein, bactericidal/permeability-increasing protein, even after several hours of stimulation. These results indicate that continuous or repeated exposure to LPS is required to maintain the expression of inflammatory genes and that the activated state is rapidly reversed with LPS neutralization.