Differential regulation of interleukin-6, macrophage inflammatory protein-1, and JE/MCP-1 cytokine expression in macrophage cell lines.

Activated macrophages produce a number of proinflammatory cytokines including IL-6, JE, MIP-1 alpha and MIP-1 beta. The induction requirements for production of either IL-6 or the MIP-1 related inflammatory proteins (MIP-1 alpha, MIP-1 beta, and JE) have been analyzed independently using fibroblasts, monocytes, or endothelial cells. However, little is known about the regulation of these cytokines in macrophages. Since activated macrophages produce prostaglandins (PGE2) which may participate in the autoregulation of cytokine production by stimulation of adenylate cyclase and the induction of cAMP-dependent signal pathways, we determined the effects of PGE on the production of IL-6 and MIP-1-related proteins. Murine macrophage cell lines were incubated with PGE1, PGE2, cholera toxin, or dibutyryl cAMP in the presence of absence suboptimal doses of LPS. Pharmacologic agents alone did not induce IL-6 production but incubation of macrophages with combinations of adenylate cyclase stimulators and LPS or dcAMP and LPS led to the dose-dependent enhancement of IL-6 secretion and mRNA expression. In contrast, PGE1 inhibits LPS-induced JE, MIP-1 alpha, and MIP-1 beta mRNA expression and this inhibition is partially dependent on a cAMP-mediated pathway of signal transduction. In previous work we demonstrated that IFN-gamma and PMA do not stimulate the production of IL-6 by macrophages. Here we show that incubation of macrophages with either IFN-gamma or PMA induces the expression of JE, MIP-1 alpha and MIP-1 beta mRNA expression. JE mRNA expression is much more responsive to the stimulatory effects of IFN-gamma than are the MIP-1 genes. Finally, PGE inhibits PMA and IFN-gamma-induced JE and MIP-1-related mRNA expression.     

Immunolocalization of CXC chemokine and recruitment of polymorphonuclear leukocytes in the rat molar periodontal tissue after topical application of lipopolysaccharide

This study investigated the recruitment of polymorphonuclear leukocytes (PMNs) and the immunolocalization of CXC chemokines, including macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant-2 (CINC-2) in rat periodontal tissue after topical application of lipopolysaccharide (LPS; 5 mg/ml) from Escherichia coli into the rat molar gingival sulcus. In normal periodontal tissues, a small number of MIP-2- and CINC-2-positive cells were seen in junctional epithelium (JE), especially in its coronal half. After topical application of LPS, a prominent increase of MIP-2- and CINC-2-positive JE cells was observed. Almost all JE cells strongly expressed them at day 1 and day 2, and then the number of chemokine-positive cells returned to normal at day 7. Corresponding to these chemokine expressions, LPS application induced a significant increase in the number of PMNs in the sub-JE area from 1 h to 2 days and a significant increase in JE area from 3 h to 5 days, indicating a dynamic flow of PMNs from the sub-JE area into JE. These findings indicated that JE cells produced MIP-2 and CINC-2 in response to LPS stimulation and suggested that MIP-2 and CINC-2 may be responsible for PMN migration toward the periodontal pathogen and may play an important role in the initiation of inflammation and subsequent periodontal tissue destruction.     

Short- and long-term effects of IL-1 and TNF antagonists on periodontal wound healing

The present study tested the effects of local injection of IL-1 and TNF soluble receptors on a periodontal wound-healing model in nonhuman primates. In this model, periodontal lesions were developed for 16 wk, followed by open flap surgery. Starting at the time of surgery, groups of animals received localized injections of both soluble cytokine receptors or else PBS three times per week for 3, 14, or 35 days. Periodontal wound healing was analyzed for each group at the end of the treatment regimen. Fourteen days after surgery, a significant decrease was observed between the animals treated with soluble receptors and the untreated group with respect to recruitment of inflammatory cells in deep gingival connective tissue. Concurrent apoptosis of inflammatory cells in those tissues increased significantly in treated animals compared with untreated animals. All other outcome parameters of periodontal wound healing were likewise significantly improved in treated animals compared with untreated animals. In marked contrast, however, 35 days after surgery, there was a significant increase in the number of inflammatory cells that had infiltrated into deep gingival connective tissue in treated compared with untreated animals. Outcome parameters of periodontal wound healing worsened in treated animals when compared with untreated. These results indicate that proinflammatory cytokines may play different functional roles in early vs late phases of periodontal wound healing. Short-term blockade of IL-1 and TNF may facilitate periodontal wound healing, whereas prolonged blockade may have adverse effects.     

Visfatin regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway

Periodontitis is a widespread chronic infectious-inflammatory disease associated with multiple systemic diseases. Visfatin is an adipokine-enzyme that can be locally produced by human periodontal ligament cells (hPDLCs) and human gingival fibroblasts (hGFs). It can upregulate proinflammatory cytokines and matrix metalloproteinases (MMPs) in various types of cells. However, the effects of visfatin on healthy and inflammatory human periodontal cells as well as the underlying molecular mechanisms remain unclear. This study firstly demonstrated visfatin expression was highly elevated in inflamed human gingiva and Pg LPS-treated hPDLCs. Moreover, recombinant visfatin significantly upregulated the expression of proinflammatory cytokines (TNF-α, IL-1β and IL-6) and prodegradative factors (EMPPRIN, MMP1, MMP3 and MMP13) in hPDLCs. Next, we found the levels of proinflammatory and prodegradative cytokines were significantly increased in visfatin-overexpressing hPDLCs, and decreased in visfatin-silencing inflammatory hGFs (iGFs) when treated with Pg LPS. In the absence of Pg LPS, visfatin silencing failed to affect the expression of these factors in iGFs, and overexpression of visfatin upregulated MMPs but no other factors in hPDLCs. Furthermore, marked NF-κB pathway activation with increased phosphorylation of p65 was observed in visfatin-overexpressing hPDLCs. BAY11-7082, a specific inhibitor of NF-κB, partially reversed the upregulation proinflammatory and prodegradative factors induced by visfatin overexpression. Taken together, this study showed that visfatin critically regulates Pg LPS-induced proinflammatory/prodegradative effects in healthy and inflammatory periodontal cells partially via NF-κB pathway. The findings suggest that visfatin is closely involved in the development of periodontitis, and may serve as a promising novel biomarker and therapeutic target for periodontitis management.     

Synergistic Anti-Inflammatory Activity of the Antimicrobial Peptides Human Beta-Defensin-3 (hBD-3) and Cathelicidin (LL-37) in a Three-Dimensional Co-Culture Model of Gingival Epithelial Cells and Fibroblasts

Given the spread of antibiotic resistance in bacterial pathogens, antimicrobial peptides that can also modulate the immune response may be a novel approach for effectively controlling periodontal infections. In the present study, we used a three-dimensional (3D) co-culture model of gingival epithelial cells and fibroblasts stimulated with Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS) to investigate the anti-inflammatory properties of human beta-defensin-3 (hBD-3) and cathelicidin (LL-37) and to determine whether these antimicrobial peptides can act in synergy. The 3D co-culture model composed of gingival fibroblasts embedded in a collagen matrix overlaid with gingival epithelial cells had a synergistic effect with respect to the secretion of IL-6 and IL-8 in response to LPS stimulation compared to fibroblasts and epithelial cells alone. The 3D co-culture model was stimulated with non-cytotoxic concentrations of hBD-3 (10 and 20 µM) and LL-37 (0.1 and 0.2 µM) individually and in combination in the presence of A. actinomycetemcomitans LPS. A multiplex ELISA assay was used to quantify the secretion of 41 different cytokines. hBD-3 and LL-37 acted in synergy to reduce the secretion of GRO-alpha, G-CSF, IP-10, IL-6, and MCP-1, but only had an additive effect on reducing the secretion of IL-8 in response to A. actinomycetemcomitans LPS stimulation. The present study showed that hBD-3 acted in synergy with LL-37 to reduce the secretion of cytokines by an LPS-stimulated 3D model of gingival mucosa. This combination of antimicrobial peptides thus shows promising potential as an adjunctive therapy for treating inflammatory periodontitis.     

Lipopolysaccharide-induced IL-1 beta production by human uterine macrophages up-regulates uterine epithelial cell expression of human beta-defensin 2

The uterine endometrium coordinates a wide spectrum of physiologic and immunologic functions, including endometrial receptivity and implantation as well as defense against sexually transmitted pathogens. Macrophages and epithelial cells cooperatively mediate innate host defense against bacterial invasion through the generation of immunologic effectors, including cytokines and antimicrobial peptides. In this study, we demonstrate that stimulation of peripheral blood monocytes and uterine macrophages with bacterial LPS induces the production of biologically active proinflammatory IL-1beta. High doses of estradiol enhance LPS-induced IL-1beta expression in an estrogen receptor-dependent manner. Furthermore, both peripheral blood monocyte- and uterine macrophage-derived IL-1beta induce secretion of antimicrobial human beta-defensin 2 by uterine epithelial cells. These data indicate dynamic immunologic interaction between uterine macrophages and epithelial cells and implicate a role for estradiol in the modulation of the immune response.     

Interleukin-1 superfamily member,interleukin-33, in periodontal diseases

Interleukin (IL) -33 is a nuclear protein that is released from damaged cells and acts as an alarmin. We investigated the expression of IL-33 in human gingival fibroblasts after stimulation by tumor necrosis factor alpha (TNF-α). Human periodontal tissue samples were collected and fixed in phosphate-buffered 4% formalin in saline and processed to paraffin blocks. TNF-α was immunostained in samples of ten periodontitis patients and ten controls. Human gingival fibroblasts were isolated using an explant culture technique. The influence of TNF-α on IL-33 in gingival fibroblasts was analyzed using enzyme-linked immunosorbent assay (ELISA). The number of TNF-α positive cells was significantly greater in periodontitis samples than in controls. TNF-α was located mainly in macrophage- and fibroblast-like cells, vascular endothelial cells and epithelial cells. Analysis of IL-33 expression in cell culture lysates showed that TNF-α induced IL-33 in cultured gingival fibroblasts. Periodontitis samples are characterized by Th2 cell dominance, which has been linked to anti-inflammatory responses and periodontal repair. TNF-α-induced IL-33 may link inflammation directly to the IL-33-dependent stimulation of Th2 cytokine producing cells and participate in the induction of lymphocytes, which results in protective, anti-inflammatory and reparative responses.     

Lipopolysaccharide promotes the survival of osteoclasts via Toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages

Lipopolysaccharide is a pathogen that causes inflammatory bone loss. Monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNF-alpha, and IL-6 in response to LPS. We examined the effects of LPS on the function of osteoclasts formed in vitro in comparison with its effect on bone marrow macrophages, osteoclast precursors. Both osteoclasts and bone marrow macrophages expressed mRNA of Toll-like receptor 4 (TLR4) and CD14, components of the LPS receptor system. LPS induced rapid degradation of I-kappaB in osteoclasts, and stimulated the survival of osteoclasts. LPS failed to support the survival of osteoclasts derived from C3H/HeJ mice, which possess a missense mutation in the TLR4 gene. The LPS-promoted survival of osteoclasts was not mediated by any of the cytokines known to prolong the survival of osteoclasts, such as IL-1beta, TNF-alpha, and receptor activator of NF-kappaB ligand. LPS stimulated the production of proinflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in bone marrow macrophages and peritoneal macrophages, but not in osteoclasts. These results indicate that osteoclasts respond to LPS through TLR4, but the characteristics of osteoclasts are quite different from those of their precursors, macrophages, in terms of proinflammatory cytokine production in response to LPS.     

Inhibition of Inducible Nitric Oxide Synthase Expression by Endothelin in Rat Glial Cells Prepared from the Neonatal Rat Brain

Abstract: In primary cultured rat glial cells, a combination of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) stimulates production of nitrite via expression of the inducible form of nitric oxide synthase (iNOS). In these cells, simultaneous addition of endothelin (ET) decreased iNOS expression and nitrite accumulation induced by TNF-α/IL-1β. The inhibitory effect of ET on TNF-α/IL-1β-stimulated iNOS expression appears to be mediated by ET_B receptors, because (1) both ET-1 and ET-3 inhibited the effects of TNF-α/IL-1β on iNOS expression and nitrite accumulation, (2) a selective ET_B receptor agonist, Suc-[Glu⁹,Ala^11,15]-ET-1 (8–21) (IRL1620), decreased the effects of TNF-α/IL-1β, and (3) a selective ET_B receptor antagonist, N-cis -2,6-dimethylpiperidinocarbonyl- l -γ-methylleucyl- d -1-methoxycarbonyltryptophanyl- d -norleucine, abolished the inhibitory effects of ETs and IRL1620. Incubation of glial cells with lipopolysaccharide (LPS) caused an increase in iNOS expression. Simultaneous addition of ET-3 decreased the effects of LPS (10 and 100 ng/ml) on iNOS expression. Furthermore, cyclic AMP-elevating agents (dibutyryl cyclic AMP and forskolin) inhibited TNF-α/IL-1β-induced and LPS-induced iNOS expression and nitrite accumulation. These findings suggest that ETs can decrease TNF-α/IL-1β-induced and LPS-induced iNOS expression via ET_B receptors and that cyclic AMP may be involved in this process.     

Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin

Together with macrophages and dendritic cells, mast cells have recently been shown to interact with certain pathogenic bacteria and present microbial antigens to the immune system. We show here that Bordetella pertussis can adhere to and be phagocytosed by mast cells. In addition, mast cells are able to process and present B. pertussis antigens to T lymphocytes. Furthermore, exposure of mast cells to B. pertussis induced the release of the proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). The release of IL-6 was strongly reduced by pertussis toxin expressed by B. pertussis . The production of IL-10, but not that of IL-4, by mast cells was also inhibited by pertussis toxin. Depletion of mast cells in vivo resulted in significant reduction of early TNF-α production in bronchoalveolar lavage (BAL) fluids of B. pertussis -infected mice. These data suggest that mast cells may play a role in the induction of immune responses against B. pertussis through the release of cytokines, especially TNF-α.     

Gingival and dermal fibroblasts produce interleukin-1 beta converting enzyme and interleukin-1 beta but not interleukin-18 even after stimulation with lipopolysaccharide

Epithelial cells play a critical role in periodontal disease through the secretion of pro-inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18). However, the role played by fibroblasts is still unclear. The rationale of this study was to throw light on the role of gingival fibroblasts in periodontal disease. We thus investigated the expression of IL-1 beta, IL-18, and ICE mRNA and the secretion of the corresponding proteins by human normal gingival fibroblasts before and after stimulation with lipopolysaccharide (LPS) from Porphyromonas gingivalis and Escherichia coli. IL-1 beta, IL-18, and ICE mRNA expression was evaluated by RT-PCR. Proteins were analyzed by Western blot and ELISA. We demonstrated that gingival fibroblasts expressed ICE mRNA. Basal expression of ICE was modulated following cell stimulation with lipopolysaccharide (5 mug/ml). However, gingival fibroblasts expressed low levels of IL-1 beta mRNA. The expression was potentiated by LPS. The expression of IL-1 beta mRNA was followed by the secretion of IL-1 beta but not IL-18 protein. Our study suggests that fibroblasts may be involved in the defense against infections via an IL-1 beta-mediated but not an IL-18-mediated mechanism.     

Pro-atherogenic lung and oral pathogens induce an inflammatory response in human and mouse mast cells

A broad variety of microbes are present in atherosclerotic plaques and chronic bacterial infection increases the risk of atherosclerosis by mechanisms that have remained vague. One possible mechanism is that bacteria or bacterial products activate plaque mast cells that are known to participate in the pathogenesis of atherosclerosis. Here, we show by real-time PCR analysis and ELISA that Chlamydia pneumoniae (Cpn) and a periodontal pathogen, Aggregatibacter actinomycetemcomitans (Aa), both induce a time and concentration-dependent expression and secretion of interleukin 8 (IL-8), tumour necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) by cultured human peripheral blood-derived mast cells, but not anti-inflammatory molecules, such as IL-10 or transforming growth factor β1 (TGF-β1). The IL-8 and MCP-1 responses were immediate, whereas the onset of TNF-α secretion was delayed. The Cpn-mediated pro-inflammatory effect was attenuated when the bacteria were inactivated by UV-treatment. Human monocyte-derived macrophages that were pre-infected with Cpn also induced a significant pro-inflammatory response in human mast cells, both in cocultures and when preconditioned media from Cpn-infected macrophages were used. Intranasal and intravenous administration of live Cpn and Aa, respectively induced an accumulation of activated mast cells in the aortic sinus of apolipoprotein E-deficient mice, however, with varying responses in the systemic levels of lipopolysaccharide (LPS) and TNF-α. Pro-atherogenic Cpn and Aa induce a pro-inflammatory response in cultured human connective tissue-type mast cells and activation of mouse aortic mast cells in vivo .     

Essential roles of high-mobility group box 1 in the development of murine colitis and colitis-associated cancer

High-mobility group box 1 (HMGB1) is a nuclear factor released extracellularly as a proinflammatory cytokine. We measured the HMGB1 concentration in the sera of mice with chemically induced colitis (DSS; dextran sulfate sodium salt) and found a marked increase. Inhibition of HMGB1 by neutralizing anti-HMGB1 antibody resulted in reduced inflammation in DSS-treated colons. In macrophages, HMGB1 induces several proinflammatory cytokines, such as IL-6, which are regulated by NF-kappaB activation. Two putative sources of HMGB1 were explored: in one, bacterial factors induce HMGB1 secretion from macrophages and in the other, necrotic epithelial cells directly release HMGB1. LPS induced a small amount of HMGB1 in macrophages, but macrophages incubated with supernatant prepared from necrotic cells and containing large amounts of HMGB1 activated NF-kappaB and induced IL-6. Using the colitis-associated cancer model, we demonstrated that neutralizing anti-HMGB1 antibody decreases tumor incidence and size. These observations suggest that HMGB1 is a potentially useful target for IBD treatment and the prevention of colitis-associated cancer.     

Differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type II epithelial cells and macrophages

Bacterial colonization is a secondary feature of many lung disorders associated with elevated cytokine levels and increased leukocyte recruitment. We hypothesized that, alongside macrophages, the epithelium would be an important source of these mediators. We investigated the effect of LPS (0, 10, 100, and 1000 ng/ml LPS, up to 24 h) on primary human lung macrophages and alveolar type II epithelial cells (ATII; isolated from resected lung tissue). Although macrophages produced higher levels of the cytokines TNF-alpha and IL-1beta (p < 0.0001), ATII cells produced higher levels of chemokines MCP-1, IL-8, and growth-related oncogene alpha (p < 0.001), in a time- and concentration-dependent manner. Macrophage (but not ATII cell) responses to LPS required activation of ERK1/2 and p38 MAPK signaling cascades; phosphorylated ERK1/2 was constitutively up-regulated in ATII cells. Blocking Abs to TNF-alpha and IL-1beta during LPS exposure showed that ATII cell (not macrophage) MCP-1 release depended on the autocrine effects of IL-1beta and TNF-alpha (p < 0.003, 24 h). ATII cell release of IL-6 depended on autocrine effects of TNF-alpha (p < 0.006, 24 h). Macrophage IL-6 release was most effectively inhibited when both TNF-alpha and IL-1beta were blocked (p < 0.03, 24 h). Conditioned media from ATII cells stimulated more leukocyte migration in vitro than conditioned media from macrophages (p < 0.0002). These results show differential activation of cytokine and chemokine release by ATII cells and macrophages following LPS exposure. Activated alveolar epithelium is an important source of chemokines that orchestrate leukocyte migration to the peripheral lung; early release of TNF-alpha and IL-1beta by stimulated macrophages may contribute to alveolar epithelial cell activation and chemokine production.     

Intraperitoneal injection of synthetic bacterial lipopeptides does not cause a rise in circulating inflammatory cytokines

Abstract The production of TNF-α, IL-1 and IL-6 was measured in mice after i.p. injections of the synthetic bacterial lipopeptide Pam₃Cys-Ala-Gly, a potent macrophage and B cell activator in vitro. Only minor amounts of IL-6 and no TNF-α were detectable in the serum of mice injected with 10 or 100 μg of Pam₃Cys-Ala-Gly. Lipopeptide concentrations up to 1000 μg failed to induce IL-1, and TNF-α production and serum IL-6 levels were only slightly elevated. In contrast to Pam₃Cys-Ala-Gly, i.p. injections of LPS were accompanied by high levels of the inflammatory cytokines TNF-α, IL-1 and IL-6 suggesting fundamental differences of the mode of action of these two substances when applied in vivo.     

Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus

Inflammation is a hallmark of brucellosis. Although Brucella abortus, one of the disease's etiologic agents, possesses cytokine-stimulatory properties, the mechanism by which this bacterium triggers a proinflammatory response is not known. We examined the mechanism whereby heat-killed B. abortus (HKBA), as well as its LPS, induces production of inflammatory cytokines in monocytes/macrophages. Polymyxin B, a specific inhibitor of LPS activity, did not inhibit the production of TNF-alpha- and IL-6-induced HKBA in the human monocytic cell line THP-1. HKBA induced the production of these cytokines in peritoneal macrophages of both C3H/HeJ and C3H/HeN mice, whereas B. abortus LPS only stimulated cells from C3H/HeN mice. Anti-TLR2 Ab, but not anti-TLR4 Ab, blocked HKBA-mediated TNF-alpha and IL-6 production in THP-1 cells. Because bacterial lipoproteins, a TLR2 ligand, have potent inherent stimulatory properties, we investigated the capacity of two B. abortus lipoproteins, outer membrane protein 19 (Omp19) and Omp16, to elicit a proinflammatory response. Lipidated (L)-Omp16 and L-Omp19, but not their unlipidated forms, induced the secretion of TNF-alpha, IL-6, IL-10, and IL-12 in a time- and dose-dependent fashion. Preincubation of THP-1 cells with anti-TLR2 Ab blocked L-Omp19-mediated TNF-alpha and IL-6 production. Together, these results entail a mechanism whereby B. abortus can stimulate cells from the innate immune system and induce cytokine-mediated inflammation in brucellosis. We submit that LPS is not the cause of inflammation in brucellosis; rather, lipoproteins of this organism trigger the production of proinflammatory cytokines, and TLR2 is involved in this process.     

Interleukin 1beta-secreting cells in inflamed gingival tissue of adult periodontitis patients.

Interleukin 1beta (IL-1beta) is a cytokine with a wide range of biological activities. It is produced by various cell types including macrophages, fibroblasts, and neutrophils. The inflammatory responses mediated by IL-1beta play an important role in periodontal tissue destruction. The purposes of this study were: (1) to determine the location of IL-1beta in inflamed human gingival tissues by the immunofluorescence method; and (2) to correlate this location to the concomitant presence of macrophage or neutrophils by immunohistochemistry. Five patients with moderate to advanced adult periodontitis receiving periodontal phase I therapy were included in this study. One month after phase I therapy, 15 sites with a probing pocket depth >/=5 mm and gingivitis index >/=1 were arranged for modified Widman flap operation. Another three sites with a probing pocket depth 相似文献