Stimulated IL-6 Production Research Articles

Haemophilus parainfluenzae infection of respiratory mucosa

The pathogenicity of Haemophilus parainfluenzae ( Hpi) in the respiratory tract is unclear, in contrast to the accepted pathogenicity of its close relative non-typable H. influenzae. We have investigated the interaction of two Hpi isolates with the mucosa of adenoid and bronchial tissue organ cultures. The adherence of bacteria to the mucosa of organ cultures, the effect of broth culture filtrates on human nasal epithelium, and interleukin (IL)-8 production by A549 cell cultures was investigated. Hpi 4846 adhered infrequently in clusters of pleomorphic cocco-bacilli to areas of epithelial damage, mucus and unciliated cells in adenoid organ culture experiments at 24 h, but not bronchial mucosa. Hpi 3698 was seen in only one adenoid and no bronchial organ cultures at 24 h. In separate experiments, Hpi 3698 was cleared more rapidly from the centre of the adenoid organ culture and was not cultured at 24 h. Although not adhering to the mucosa at 24 h, Hpi 3698, but not Hpi 4846, caused an increase in the amount of epithelial damage in both types of organ culture. Broth culture filtrates of both strains caused immediate slowing of ciliary beat frequency that progressed, and disrupted epithelial integrity. Dialysed culture filtrates of both strains stimulated IL-8 production by A549 cells, with the culture filtrate of Hpi 3698 being most potent. We conclude that two strains of Hpi varied in their adherence to adenoid tissue, and neither adhered to bronchial tissue. These results lead us to speculate that Hpi is only likely to be a pathogen in the lower respiratory tract when impaired airway defences delay bacterial clearance.

An intestinal epithelial lectin, galectin-4, specifically stimulates IL-6 production of colitic CD4+ T cells and contributes to the perpetuation of colitis

An intestinal epithelial lectin, galectin-4, specifically stimulates IL-6 production of colitic CD4+ T cells and contributes to the perpetuation of colitis

Dendritic cells and measles virus infection.

Measles is a major cause of childhood mortality in developing countries which is mainly attributed to the ability of measles virus (MV) to suppress general immune responses. Paradoxically, virus-specific immunity is efficiently induced, which leads to viral clearance from the host and confers long-lasting protection against reinfection. As sensitisers of pathogen encounter and instructors of the adaptive immune response, dendritic cells (DCs) may play a decisive role in the induction and quality of the MV-specific immune activation. The ability of MV wild-type strains in particular to infect DCs in vitro is dearly established, and the receptor binding haemagglutinin protein of these viruses essentially determines this particular tropism. DC maturation as induced early after MV infection is likely to be of crucial importance for the induction of MV-specific immunity. DCs may, however, be instrumental in MV-induced immunosuppression. (1) T cell depletion could be brought about by DC-T cell fusion or TRAIL-mediated induction of apoptosis. (2) Inhibition of stimulated IL-12 production from MV-infected DCs might affect T cell responses in qualitative terms in favouring Th2 and suppressing Th1 responses. (3) The viral glycoprotein complex expressed at high levels on infected DCs late in infection is able to directly inhibit T cell proliferation by surface contact-dependent negative signalling. This most likely accounts for the failure of infected DC cultures to stimulate allogeneic and inhibit mitogen-stimulated T cell proliferation in vitro and the pronounced proliferative unresponsiveness of T cell ex vivo to polyclonal and antigen-specific stimulation which is a central finding of MV-induced immunosuppression.

NCX-1015, a nitric-oxide derivative of prednisolone, enhances regulatory T cells in the lamina propria and protects against 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice.

NCX-1015 is a nitric oxide (NO)-releasing derivative of prednisolone. In this study we show NCX-1015 protects mice against the S. A. development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS). The beneficial effect of NCX-1015 was reflected in increased survival rates, improvement of macroscopic and histologic scores, a decrease in the mucosal content of T helper cell type 1 cytokines (protein and mRNA), and diminished myeloperoxidase activity in the colon. In contrast to its NO derivative, only very high doses of prednisolone were effective in reproducing these beneficial effects. NCX-1015 was 10- to 20-fold more potent than the parent compound in inhibiting IFN-gamma secretion by lamina propria mononuclear cells. Protection against developing colitis correlated with inhibition of nuclear translocation of p65Rel A in these cells. In vivo treatment with NCX-1015 potently stimulated IL-10 production, suggesting that the NO steroid induces a regulatory subset of T cells that negatively modulates intestinal inflammation.

Hyperthermia-induced heat shock activates the transcription factor c/EBP-beta and augments IL-6 production in human intestinal epithelial cells.

Interleukin (IL)-6 production is increased in gut mucosa during sepsis and endotoxemia. The heat shock response augments IL-6 production under these conditions, but the mechanism is not known. We hypothesized that heat shock stimulates IL-6 production in enterocytes by increasing expression and activity of the transcription factor C/EBB. Cultured Caco-2 cells, a human intestinal epithelial cell line, underwent induction of the heat shock response by hyperthermia (43 degrees C for 1 hour). Other cells were kept at 37 degrees C. Cells were then treated with 0.5 ng/mL human recombinant IL-1beta for 4 hours. C/EBP-beta and delta DNA binding activity was determined by electrophoretic mobility shift assay and supershift analysis. In additional experiments, Caco-2 cells were transfected with expression plasmids for C/EBP-beta and delta, after which cells were subjected to hyperthermia and treatment with IL-1beta. C/EBP-beta, but not delta, protein levels and DNA binding activity were increased in Caco-2 cells expressing the heat shock response. Induction of the heat shock response augmented IL-6 production in IL-1beta-treated cells overexpressing C/EBP-beta, but not delta. Increased IL-6 production in IL-1beta-treated enterocytes expressing the heat shock response might be caused by upregulated expression and activity of CIEBP-beta. Because recent studies suggest that IL-6 might be an antiinflammatory cytokine and might exert protective effects in gut mucosa and enterocytes, understanding mechanisms by which the heat shock response augments IL-6 production might have important clinical implications.

Extracellular adenine nucleotides modulate cytokine production by human monocyte-derived dendritic cells: dual effect on IL-12 and stimulation of IL-10.

To clarify the functional consequences of adenine nucleotides action on human monocyte-derived dendritic cells (DC), we have systematically compared the effects of adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), an ATP analog active on the P2Y(11) receptor, on the responses to three DC stimuli, TNF-alpha, LPS, sCD40L, tested at various concentrations, using two different IL-12 assays. We observed that ATPgammaS potentiated the IL-12p40 release induced by TNF-alpha, but also by lipopolysaccharides (LPS) and soluble CD40 ligand (sCD40L). This potentiation was observed as long as the IL-12p40 concentration under agonist stimulation remained below a threshold value close to 10 ng/ml; inhibition was observed above this value. The combinations ATPgammaS-TNF-alpha and ATPgammaS-sCD40L were unable to induce detectable bioactive IL-12p70 production and at concentrations of LPS that induced a significant stimulation of IL-12p70, the effect of ATPgammaS was purely inhibitory. Our results also show that ATPgammaS synergized with LPS and sCD40L, but not TNF-alpha, to stimulate IL-10 production. In conclusion, we have clarified the discrepancies in the literature concerning the action of adenine nucleotides on DC and our study supports the concept that, like prostaglandin E(2) and other agents increasing cyclic AMP, they favor either a Th2 response or tolerance.

Single treatment (hormonal imprinting) of newborn rats with serotonin increases the serotonin content of cells in adults.

Hormonal imprinting takes place perinatally at the first encounter between the hormone and its target receptor, causing the finishment of the maturation of receptor-signal transduction system. In the presence of an excess of the target hormone or related molecules faulty imprinting develops with life-long consequences. In earlier experiments single neonatal treatment with minute dose of IL-6 caused also prolonged stimulation of IL-6 production. In the present experiment newborn female and male rats were treated with 20 microg serotonin (hormonal imprinting) and were studied for serotonin content of different cell types in adult age. Serotonin content was measured by flow cytometry and its localization was determined by confocal microscopy. Serotonin content was detected in white blood cells (lymphocytes, monocytes and granulocytes); in lymphocytes, monocytes (macrophages), granulocytes and mast cells of peritoneal fluid and thymic lymphocytes. Serotonin was present in all cell types of control animals studied. Serotonin content extremely elevated in the white blood cells and also increased in the peritoneal cells of neonatally treated female animals. There was no elevation in thymic lymphocytes. The mean values of male animals remained at the control level. The experiments call attention to the life-long effect of the perinatal hormonal imprinting manifested presently in the elevation of serotonin content and point to the gender differences of serotonin imprinting. Considering the role of serotonin in mood and psychiatric diseases, the observations could have some clinical importance.

Interleukin-1β stimulates interleukin-8 production and gene expression in synovial cells from human temporomandibular joint

Purpose: The aims of the present study were to isolate and characterize cultured synovial cells from human temporomandibular joint (TMJ) specimens and to investigate the effect of interleukin (IL)-1β on IL-8 production and gene expression in those cells. Materials and Methods: Synovial cells (HTS cells) were isolated from TMJ synovial tissues using an outgrowth method and then primary cultured. The cells were examined for cell-specific markers of fibroblast, macrophage, and dendritic cells using immunocytochemistry. HTS cells were then treated with IL-1β, and amounts of IL-8 were measured by enzyme-linked immunosorbent assay. IL-8 production and expression were also investigated using immunocytochemistry and a reverse transcription–polymerase chain reaction method. Results: HTS cells were positive for the fibroblast-specific markers, such as vimentin and propyl 4-hydroxylase. The macrophage or dendritic cell markers and HLA class II antigen were negative. Furthermore, IL-1β enhanced IL-8 production in HTS cells in a time- and dose-dependent manner and stimulated IL-8 gene expression. Conclusions: HTS cells may provide important advantages for studies of the cellular and molecular mechanisms in the TMJ. In addition, we found that IL-1β stimulated IL-8 production through an increase in IL-8 gene expression in HTS cells, which may be associated with the increase of infiltrating inflammatory cells seen in the synovial membrane of TMJ disorders. © 2002 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 60:741-747, 2002

Can altered production of interleukin-1β, interleukin-6, transforming growth factor-β and prostaglandin E2 by isolated human subchondral osteoblasts identify two subgroups of osteoarthritic patients

Objective To determine the capacity of human subchondral osteoarthritic osteoblasts (Ob) to produce interleukin (IL)-1β, IL-6, transforming growth factor-β (TGF-β) and prostaglandin E2 (PGE2), and determine if a relationship exists between IL-1β, TGF-β, PGE2 and IL-6 production.Methods We measured the abundance of IL-1β, IL-6, TGF-β and PGE2 using very sensitive ELISA in conditioned-media of human primary subchondral Ob from normal individuals and osteoarthritic patients. Selective inhibition of IL-6 or IL-6 receptor signaling was performed to determine its effect on PGE2 production whereas the inhibiton of PGE2 production was performed to determine its effect on IL-6 production. The expression of bone cell markers and urokinase plasminogen activator (uPA) activity was also determined.Results Osteoarthritic Ob produced all these factors with greater variability than normal cells. Interestingly, the production of IL-6 and PGE2 by osteoarthritic Ob separated patients into two subgroups, those whose Ob produced levels comparable to normal (low producers) and those whose Ob produced higher levels (high producers). In those cells classified as high osteoarthritic Ob, PGE2 and IL-6 levels were increased two- to three-fold and five- to six-fold, respectively, compared with normal. In contrast, while using their IL-6 and PGE2 production to separate osteoarthritic Ob into low and high producers, we found that IL-1β levels were similar in normal and all osteoarthritic Ob. Using the same criteria, TGF-β levels were increased in all osteoarthritic Ob compared with normal. Reducing PGE2 synthesis by Indomethacin [a cyclo-oxygenase (COX) -1 and -2 inhibitor] reduced IL-6 levels in all osteoarthritic Ob, whereas Naproxen (a more selective COX-2 inhbitor) reduced PGE2 and IL-6 levels only in the high osteoarthritic group. Conversely, PGE2 addition to osteoarthritic Ob enhanced IL-6 production in both groups. Moreover, the addition of parathyroid hormone also stimulated IL-6 production to similar normal levels in both osteoarthritic groups. In contrast, using an antibody against IL-6 or IL-6 receptors did not reduce PGE2 levels in either group. The evaluation of alkaline phosphatase activity, osteocalcin release, collagen type I and uPA activity in osteoarthritic Ob failed to show any differences between these cells regardless to which subgroup they were assigned.Conclusions These results indicate that IL-6 and PGE2 production by subchondral Ob can discriminate two subgroups of osteoarthritic patients that cannot otherwise be separated by their expression of cell markers, and that endogenous PGE2 levels influence IL-6 synthesis in osteoarthritic Ob. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

Production of interleukin-6 by skeletal myotubes: role of reactive oxygen species.

In the present study we have tested the ability of reactive oxygen species (ROS) to stimulate the production of interleukin (IL)- 6 from skeletal myocytes. Differentiated C2C12 murine skeletal muscle cells (myotubes) exposed to pyrogallol (PYR), xanthine/ xanthine-oxidase (X/XO), or H(2)O(2) for 24 h exhibited a concentration-dependent increase in IL-6 production. Unlike myotubes, incubation of myoblasts and endothelial cells with X/XO or PYR did not result in increased IL-6 release. In myotubes, superoxide dismutase and catalase blocked the ROS-induced IL-6 release. Exposure of myotubes to H(2)O(2) increased steady-state IL-6 mRNA levels, and pretreatment of myotubes with actinomycin D or cycloheximide abolished the ROS-induced IL-6 production. In addition, pretreatment of cells with N-acetyl-cysteine blocked tumor necrosis factor (TNF)-alpha-induced IL-6 release, suggesting that endogenously produced ROS participate in IL-6 production. Myotubes stimulated with H(2)O(2) exhibited increased I kappa B-alpha phosphorylation and degradation, and treatment of C2C12 with ROS-generating agents increased activator protein (AP)-1 and nuclear factor (NF)-kappa B-dependent promoter activity. Finally, preincubation of myotubes with the pharmacologic inhibitor of NF-kappa B, diethyldithiocarbamate, or transient transfection with an I kappa B-alpha mutant, inhibited the ROS-stimulated IL-6 release. In conclusion, ROS stimulate IL-6 production from skeletal myotubes in a manner that involves transcriptional activation of the IL-6 gene through an NF-kappa B-dependent pathway.

Proteasome inhibitors induce heat shock response and increase IL-6 expression in human intestinal epithelial cells.

In previous studies, the heat shock response, induced by hyperthermia or sodium arsenite, increased interleukin (IL)-6 production in intestinal mucosa and cultured human enterocytes. A novel way to induce the heat shock response, documented in other cell types, is treatment with proteasome inhibitors. It is not known if proteasome inhibition induces heat shock in enterocytes or influences IL-6 production. Here we tested the hypothesis that treatment of cultured Caco-2 cells, a human intestinal epithelial cell line, with proteasome inhibitors induces the heat shock response and stimulates IL-6 production. Treatment of Caco-2 cells with one of the proteasome inhibitors MG-132 or lactacystin activated the transcription factor heat shock factors (HSF)-1 and -2 and upregulated cellular levels of the 72-kDa heat shock protein HSP-72. The same treatment resulted in increased gene and protein expression of IL-6, a response that was blocked by quercetin. Additional experiments revealed that the IL-6 gene promoter contains a HSF-responsive element and that the IL-6 gene may be regulated by the heat shock response. The present results suggest that proteasome inhibition induces heat shock response and IL-6 production in enterocytes and that IL-6 may be a heat shock-responsive gene, at least under certain circumstances. The observations are important considering the multiple biological roles of IL-6, both locally in the gut mucosa and systemically, and considering recent proposals in the literature to use proteasome inhibitors in the clinical setting to induce the heat shock response.

Induction of MHC class I presentation of exogenous antigen by dendritic cells is controlled by CD4+ T cells engaging class II molecules in cholesterol-rich domains.

We investigated interactions between CD4+ T cells and dendritic cells (DC) necessary for presentation of exogenous Ag by DC to CD8+ T cells. CD4+ T cells responding to their cognate Ag presented by MHC class II molecules of DC were necessary for induction of CD8+ T cell responses to MHC class I-associated Ag, but their ability to do so depended on the manner in which class II-peptide complexes were formed. DC derived from short-term mouse bone marrow culture efficiently took up Ag encapsulated in IgG FcR-targeted liposomes and stimulated CD4+ T cell responses to Ag-derived peptides associated with class II molecules. This CD4+ T cell-DC interaction resulted in expression by the DC of complexes of class I molecules and peptides from the Ag delivered in liposomes and permitted expression of the activation marker CD69 and cytotoxic responses by naive CD8+ T cells. However, while free peptides in solution loaded onto DC class II molecules could stimulate IL-2 production by CD4+ T cells as efficiently as peptides derived from endocytosed Ag, they could not stimulate induction of cytotoxic responses by CD8+ T cells to Ag delivered in liposomes into the same DC. Signals requiring class II molecules loaded with endocytosed Ag, but not free peptide, were inhibited by methyl-beta-cyclodextrin, which depletes cell membrane cholesterol. CD4+ T cell signals thus require class II molecules in cholesterol-rich domains of DC for induction of CD8+ T cell responses to exogenous Ag by inducing DC to process this Ag for class I presentation.

Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis.

Antigen-specific T helper type 1 (Th1) cells mediate protective immunity against a range of infectious diseases, including that caused by Bordetella pertussis. Distinct T cell subtypes that secrete interleukin (IL)-10 or tumor growth factor (TGF)-β are considered to play a role in the maintenance of self-tolerance. However, the antigens recognized by these regulatory T cells in vivo have not been defined. Here we provide the first demonstration of pathogen-specific T regulatory type 1 (Tr1) cells at the clonal level and demonstrate that these cells are induced at a mucosal surface during an infection where local Th1 responses are suppressed. Tr1 clones specific for filamentous hemagglutinin (FHA) and pertactin were generated from the lungs of mice during acute infection with B. pertussis. The Tr1 clones expressed T1/ST2 and CC chemokine receptor 5, secreted high levels of IL-10, but not IL-4 or interferon (IFN)-γ, and suppressed Th1 responses against B. pertussis or an unrelated pathogen. Furthermore, FHA inhibited IL-12 and stimulated IL-10 production by dendritic cells (DCs), and these DCs directed naive T cells into the regulatory subtype. The induction of Tr1 cells after interaction of a pathogen-derived molecule with cells of the innate immune system represents a novel strategy exploited by an infectious pathogen to subvert protective immune responses in vivo.

C1q-bearing immune complexes induce IL-8 secretion in human umbilical vein endothelial cells (HUVEC) through protein tyrosine kinase- and mitogen-activated protein kinase-dependent mechanisms: evidence that the 126 kD phagocytic C1q receptor mediates immune complex activation of HUVEC.

Endothelial cells play a pivotal role in the initiation and perpetuation of inflammation. C1q, the first component of the classical pathway of complement, is a potent stimulus leading to endothelial cell activation and cytokine production. The specific cellular mechanisms through which endothelial cells are stimulated by C1q are not known. We stimulated human umbilical vein endothelial cells (HUVEC) with either monomeric C1q or C1q-bearing immune complexes (C1q-IC) in the presence or absence of inhibitors of protein tyrosine kinases (PTK) or mitogen-activated protein kinases (MAPK). C1q-IC, but not monomeric C1q, induced IL-8 production in dose- and time-dependent fashion. R3, a cross-linking monoclonal IgM antibody against the 126 kD phagocytic C1q receptor (C1qR), also stimulated IL-8 production. IL-8 mRNA accumulation was detected by Northern blot analysis within 2 h of stimulation by the immune complexes and was enhanced by the addition of cycloheximide. Secretion of IL-8 by C1q-IC stimulated HUVEC was completely blocked by the PTK inhibitor, genistein or the MAPK inhibitor, UO126. These experiments demonstrate that C1q-IC-induced production of IL-8 in HUVEC is dependent upon the activation of PTK and MAPK. These findings also support a role for the phagocytic C1qR as an important activator of HUVEC by immune complexes.

Expression of tumour necrosis factor (TNF) ligand superfamily co-stimulatory molecules CD30L, CD27L, OX40L, and 4-1BBL in murine hearts with acute myocarditis caused by Coxsackievirus B3.

Antigen-specific T-cells infiltrate the heart and play an important role in the myocardial damage involved in acute myocarditis and dilated cardiomyopathy. To investigate the roles of the co-stimulatory molecules CD30/CD30L, CD27/CD27L, OX40/OX40L, and 4-1BB/4-1BBL, which belong to the tumor necrosis factor (TNF) receptor/ligand superfamily, in the development of acute viral myocarditis, the expression of these molecules was first analysed in the hearts of mice with acute myocarditis induced by Coxsackievirus B3 (CVB3) in vivo. Secondly, the induction of these molecules was evaluated on cultured cardiac myocytes treated with interferon (IFN)-gamma and the interleukin (IL)-6 production by cultured cardiac myocytes was analysed by stimulation with monoclonal antibodies (MAbs) against these molecules in vitro. Thirdly, the effects of in vivo administration of anti-CD30L, anti-CD27L, anti-OX40L, or anti-4-1BBL MAb on the development of acute viral myocarditis were examined. CVB3-induced myocarditis resulted in the induction of CD30L and 4-1BBL on the surface of cardiac myocytes, confirmed by treatment with IFN-gamma in vitro. CD27L and OX40L were constitutively expressed on cardiac myocytes in vivo and in vitro. Anti-CD30L and anti-4-1BBL MAbs stimulated IL-6 production by cardiac myocytes in vitro. Furthermore, in vivo anti-4-1BBL MAb treatment significantly decreased the myocardial inflammation, whereas the other MAbs did not. These findings suggest that TNF ligand superfamily co-stimulatory molecules, especially 4-1BBL, play an important role in the development of acute viral myocarditis and raise the possibility that immunotherapy with anti-4-1BBL MAb may be of benefit in acute viral myocarditis.

Regulation of cytokine production in human gingival fibroblasts following treatment with nicotine and lipopolysaccharide.

Patients who smoke are at increased risk for chronic periodontitis (CP). Most studies suggest that the microbial flora in these patients is similar to that found in non-smoking CP patients. Thus, the increased risk for development of CP is not dependent on an altered microbial profile, but rather to some change in the host response to these periopathogens. There is evidence that human gingival fibroblasts (HGF) derived from diseased sites produce greater amounts of interleukin (IL)-6 and IL-8 in vitro than cells derived from healthy sites. This suggests that HGF subpopulations may be selected based upon the inflammatory milieu in which they reside. The hypothesis to be tested was that the combination of nicotine and lipopolysaccharide (LPS) could regulate HGF inflammatory mediator production. HGF cell cultures were established from explants derived from 10 patients with CP. HGF cell cultures were stimulated with 1 mM, 1 microM, or 1 nM nicotine +/- Escherichia coli or Porphyromonas gingivalis LPS. At 12, 24, or 48-hour time points, the cells were counted and the supernatant was collected for subsequent IL-6 and IL-8 determination in an enzyme-linked immunosorbent assay. At the 24-hour time point, 1 nM nicotine stimulated IL-6 production compared to control (P=0.02). E. coli LPS alone caused a 3- to 4-fold increase in IL-6 and IL-8 production, whereas P gingivalis LPS did not augment IL-6 or IL-8. A synergistic effect upregulating IL-6 was observed with combined treatment of 1 mM nicotine and E. coli LPS or P gingivalis LPS at the 24-hour time point (P<0.0005 and P=0.002, respectively). Similar effects were seen when IL-8 production was evaluated following HGF stimulation with high doses of nicotine and E. coli LPS or P gingivalis LPS. These results demonstrate that nicotine by itself can stimulate HGF IL-6 and IL-8 production. Moreover, the combination of high doses of nicotine and either E. coli or P gingivalis LPS can synergistically upregulate cytokine production. These findings support the hypothesis that a proinflammatory fibroblast phenotype may be elicited in an environment enriched with bacterial LPS and nicotine.

Medium‐chain fatty acids stimulate interleukin‐8 production in Caco‐2 cells with different mechanisms from long‐chain fatty acids1

It has been suggested that dietary fat exacerbates intestinal inflammation. We investigated the effect of fatty acids on interleukin (IL)-8 production in a human intestinal epithelial cell line (Caco-2). The cells were cultured as monolayers on microporous membranes in culture inserts. Oleic acid (OA), capric acid (CA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were applied to the apical compartment of Caco-2 cell monolayers. The concentration of IL-8 in the basolateral medium was measured by using enzyme-linked immunosorbent assay, and the expression of IL-8 mRNA was measured by using competitive reverse transcription--polymerase chain reaction. Protein kinase C inhibitors (GF109203X and calphostin C) and H-7 (a protein kinase inhibitor) were used to study the mechanisms by which IL-8 production is stimulated. Both OA and CA enhanced IL-8 production (approximately fivefold), whereas DHA and EPA did not. Both OA and CA also enhanced IL-1-induced IL-8 production. The onset of OA-induced IL-8 production was delayed compared with that of CA-induced IL-8 production. Both OA and CA enhanced IL-8 mRNA expression (approximately fivefold) after 6 and 3 h, respectively. The protein kinase inhibitor (H-7) reduced both OA- and CA-induced IL-8 production by 88.0 and 85.9%, respectively. The protein kinase C inhibitors (GF109203X and calphostin C) reduced OA-induced IL-8 production by 29.3 and 54.5%, respectively, but showed no effect on CA-induced IL-8 production. These findings suggest that not only OA but also CA stimulates IL-8 production in intestinal epithelial cells, and the mechanisms of action differ between OA and CA.

Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and activities of human defensins and cathelicidin (LL-37)

Defensins and cathelicidins are the two major families of mammalian anti-microbial proteins. They contribute to host, innate, anti-microbial defense by disrupting the integrity of the bacterial cell membrane. However, several members of the mammalian anti-microbial proteins including defensins and cathelicidins have been shown recently to have chemotactic effects on host cells. Human neutrophil alpha-defensins are chemotactic for resting, naïve CD45RA/CD4 T cells, CD8 T cells, and immature dendritic cells. Human beta-defensins are also chemotactic for immature dendritic cells but induce the migration of memory CD45RO/CD4 T cells. In contrast, cathelicidin/LL-37 is chemotactic for neutrophils, monocytes, and T cells but not for dendritic cells. Thus, these anti-microbial peptides have distinct, host-target cell spectra. The chemotactic activities of human beta-defensins and cathelicidin/LL-37 are mediated by human CC chemokine receptor 6 and formyl peptide receptor-like 1, respectively. The capacities of defensins and cathelicidins to mobilize various types of phagocytic leukocytes, immature dendritic cells, and lymphocytes, together with their other effects such as stimulating IL-8 production and mast cell degranulation, provide evidence for their participation in alerting, mobilizing, and amplifying innate and adaptive anti-microbial immunity of the host.

Synthesis of C-X-C and C-C Chemokines by Human Peritoneal Fibroblasts: Induction by Macrophage-Derived Cytokines

Leukocyte accumulation during peritonitis is believed to be controlled by chemotactic factors released by resident peritoneal macrophages or mesothelial cells. Recent data indicate, however, that in many tissues fibroblasts play a key role in mediating leukocyte recruitment. We have therefore examined human peritoneal fibroblasts (HPFBs) for the expression and regulation of C-X-C and C-C chemokines. Quiescent HPFBs secreted monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 constitutively. This release could be dose-dependently augmented with the pro-inflammatory cytokines IL-1beta and tumor necrosis factor-alpha. Stimulated IL-8 production reached a plateau within 48 hours while MCP-1 continued to accumulate throughout 96 hours. Induction of IL-8 and MCP-1 synthesis by HPFBs was also triggered by peritoneal macrophage-conditioned medium. This effect was partly related to the presence of IL-1beta as demonstrated by IL-1 receptor antagonist inhibition. Pretreatment of HPFBs with actinomycin D or puromycin dose-dependently reduced cytokine-stimulated IL-8 and MCP-1 secretion, which suggested de novo chemokine synthesis. Indeed, exposure of HPFBs to IL-1beta and tumor necrosis factor-alpha produced a significant up-regulation of IL-8 and MCP-1 mRNA. This effect was associated with the rapid induction of nuclear factor-kappaB binding activity mediated through p65 and p50 subunits, and with a transient increase in the mRNA expression for RelB and inhibitory protein kappaB-alpha proteins. These data indicate that peritoneal fibroblasts are capable of generating large quantities of chemokines under a tight control of nuclear factor-kappaB/Rel transcription factors. Thus, peritoneal fibroblast-derived chemokines may contribute to the intraperitoneal recruitment of leukocytes during peritonitis.

Prostaglandin F2alpha upregulates interleukin-6 production in human gingival fibroblasts.

Prostaglandin F2alpha (PGF2alpha) is a bioactive lipid mediator which has been suggested to be involved in the pathogenesis of periodontal disease. However, the roles of PGF2alpha in periodontal lesions are poorly understood. In the present study, we investigated the effect of PGF2alpha on interleukin (IL)-6 production in human gingival fibroblasts (HGF). PGF2alpha stimulated IL-6 production in a time- and concentration-dependent fashion. IL-1beta and tumor necrosis factor alpha (TNFalpha), proinflammatory cytokines, induced IL-6 production in a time-dependent manner, and PGF2alpha synergistically enhanced IL-6 production induced by IL-1beta and TNFalpha. IL-6 mRNA was expressed in PGF2alpha-stimulated HGF, and PGF2alpha increased IL-6 mRNA levels induced by IL-1beta and TNFalpha. Fluprostenol, a selective FP receptor agonist, could mimic PGF2alpha-induced IL-6 production. Since FP receptors are coupled to elevation of intracellular calcium and activation of protein kinase C (PKC), the mechanism of IL-6 production by PGF2alpha was investigated using TMB-8, an inhibitor of Ca2+ mobilization from intracellular stores, and calphostin C, an inhibitor of PKC. TMB-8 significantly suppressed PGF2alpha-induced IL-6 production, whereas calphostin C showed a stimulatory effect on PGF2alpha-induced IL-6 production. From these data, we suggest that PGF2alpha upregulates IL-6 production through FP receptors in HGF, that PGF2alpha synergistically enhances IL-6 production in IL-1beta- and TNFalpha-stimulated HGF, and that PGF2alpha-induced IL-6 production may be dependent on intracellular Ca2+ mobilization and be downregulated by PKC activation. PGF2alpha may be involved in the pathogenesis of periodontal disease by enhancing IL-6 levels in periodontal lesions.