Regulation of periodontal ligament cell functions by interleukin-1beta

Periodontal ligament (PDL) cells maintain the attachment of the tooth to alveolar bone. These cells reside at a site in which they are challenged frequently by bacterial products and proinflammatory cytokines, such as interleukin-1beta (IL-1beta), during infections. In our initial studies we observed that IL-1beta down-regulates the osteoblast-like characteristics of PDL cells in vitro. Therefore, we examined the functional significance of the loss of the PDL cell's osteoblast-like characteristics during inflammation. In this report we show that, during inflammation, IL-1beta can modulate the phenotypic characteristics of PDL cells to a more functionally significant lipopolysaccharide (LPS)-responsive phenotype. In a healthy periodontium PDL cells exhibit an osteoblast-like phenotype and are unresponsive to gram-negative bacterial LPS. Treatment of PDL cells with IL-1beta inhibits the expression of their osteoblast-like characteristics, as assessed by the failure to express transforming growth factor beta1 (TGF-beta1) and proteins associated with mineralization, such as alkaline phosphatase and osteocalcin. As a consequence of this IL-1beta-induced phenotypic change, PDL cells become responsive to LPS and synthesize proinflammatory cytokines. The IL-1beta-induced phenotypic changes in PDL cells were transient, as removal of IL-1beta from PDL cell cultures resulted in reacquisition of their osteoblast-like characteristics and lack of LPS responsiveness. The IL-1beta-induced phenotypic changes occurred at concentrations that are frequently observed in tissue exudates during periodontal inflammation (0.05 to 5 ng/ml). The results suggest that, during inflammation in vivo, IL-1beta may modulate PDL cell functions, allowing PDL cells to participate directly in the disease process by assuming LPS responsiveness at the expense of their normal structural properties and functions.     

Shift from anti- to proinflammatory cytokine profiles in microglia through LPS- or IFN-gamma-mediated pathways

In this study, cytokine mRNA profiles in microglia from newborn rats were detected by in situ hybridization. Under natural culture conditions, microglia expressed the immunosuppressive transforming growth factor-beta 1 (TGF-beta 1) and interleukin (IL) 10 to a greater degree than the pro-inflammatory cytokines IL-1 beta, IL-6, IL-12, interferon-gamma (IFN-gamma) and TNF-alpha. High TGF-beta 1 and IL-10 levels could reflect one mechanism for immune privilege within the CNS under physiological conditions. Stimulation of microglia with LPS or IFN gamma resulted in strong up-regulation of proinflammatory cytokines, while TGF-beta 1 and IL-10 were down-regulated. These effects of LPS or IFN-gamma are anticipated to reflect immunopathogenic processes within the CNS.     

Modulation of collagen gene expression by cytokines: stimulatory effect of transforming growth factor-beta1, with divergent effects of epidermal growth factor and tumor necrosis factor-alpha on collagen type I and collagen type IV

Transforming growth factor-beta1 (TGF-beta1) is well recognized as a potent mediator of both fibrillar (collagen type I) and basement membrane (collagen type IV) production. However, tissue injury is characterized by the concomitant expression of many cytokines and/or growth factors in addition to TGF-beta1, and the ultimate extent of extracellular-matrix (ECM) deposition may reflect the interacting effects of TGF-beta1 and these other cytokines and/or growth factors. We, therefore, sought to determine whether other cytokines and/or growth factors, known to be produced after tissue injury, are capable either alone or in combination with TGF-beta1 of modulating collagen gene expression. Collagen type I and collagen type IV gene expression was assessed in NIH-3T3 cells, a murine fibroblast-like cell line that responds to TGF-beta1, with increases in both collagen type I and collagen type IV production. TGF-beta1 coordinately induced production of collagen type IV messenger ribonucleic acid (mRNA) to a level 3.8-fold above its baseline value (p < 0.001) and collagen type I mRNA to a level 2.6-fold above its baseline value (p < 0.001). Of the other cytokines and/or growth factors tested, only epidermal growth factor (EGF) had significant effects on collagen mRNA expression. We report the novel observation that EGF significantly induced collagen type IV mRNA (3.0-fold; p < 0.001) but did not alter collagen type I mRNA expression. Platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and insulin-like growth factor-1 (IGF-1) did not alter the expression of mRNA for collagen type IV or collagen type I. Addition of TGF-beta1 to cytokine- and/or growth factor-treated cells increased both collagen type IV and collagen type I mRNA levels. However, collagen type IV mRNA levels were similar in cultures given TGF-beta1 alone and cultures given TGF-beta1 with other cytokines and/or growth factors; there were no additive, synergistic, or antagonistic effects after coadministration of TGF-beta1 and other cytokines and/or growth factors. With regard to collagen type I mRNA expression, all cytokines and/or growth factors tested, with the exception of TNF-alpha, had no effect on collagen type I mRNA levels in TGF-beta1-treated cultures. Importantly, TNF-alpha antagonized the stimulatory effect of TGF-beta1 on collagen type I mRNA levels. These observations support a dominant role for TGF-beta1 in stimulating coordinate expression of collagen type I and collagen type IV mRNAs by NIH-3T3 cells; EGF and TNF-alpha are capable of inducing divergent expression of the genes for these two types of collagen.     

PDGF-alpha receptor subunit expression down-regulated by IL-1beta in human periodontal ligament cells

The responses of cells to the distinct PDGF isoforms have been correlated directly to the relative numbers of specific PDGF receptor subunits on the cell surface. The modulation of PDGF-alpha receptor subunits, the major subunit expressed in human periodontal ligament (PDL) cells, by cytokines present in the periodontal wound site, such as interleukin-1 (IL-1), may be an important factor influencing regenerative outcomes. The purpose of the present study was to examine the effects of IL-1 beta on PDGF-alpha receptor subunit expression in human PDL cells. Primary cultures of human PDL cells were treated with IL-1 beta over a range of concentrations. We assessed PDGF-alpha receptor subunits by examining the mitogenic responses of cells to PDGF-AA, specific binding of 125I-labeled PDGF-AA, immunofluorescent analysis of PDGF-alpha receptor subunits, and PDGF-alpha receptor subunit mRNA levels using Northern blot analysis. The results demonstrate a significant concentration-dependent decrease in 3H-thymidine incorporation in response to PDGF-AA following IL-1 beta treatment (p < 0.001). This decreased response correlated directly with IL-1-induced decreases in 125I-labeled PDGF-AA binding (p < 0.01), the numbers of immunolabeled PDGF-alpha receptor subunits, and in PDGF-alpha receptor subunit mRNA levels. However, when combined with TGF-beta, IL-1 beta did not show additional down-regulation in proliferative response to PDGF-AA or PDGF-alpha receptor subunits beyond that achieved with these factors individually. These experiments identify IL-1 beta, along with TGF-beta, as significant inhibitors of PDGF stimulation in human PDL cells, acting through the down-regulation of PDGF-alpha receptor subunit expression.     

Regulated production of type I collagen and inflammatory cytokines by peripheral blood fibrocytes

We recently described a novel population of blood-borne cells, termed fibrocytes, that display a distinct cell surface phenotype (collagen+/CD13+/CD34+/CD45+), rapidly enter sites of tissue injury, and contribute to scar formation. To further characterize the role of these cells in vivo, we examined the expression of type I collagen and cytokine mRNAs by cells isolated from wound chambers implanted into mice. Five days after chamber implantation, CD34+ fibrocytes but not CD14+ monocytes or CD90+ T cells expressed mRNA for type I collagen. Fibrocytes purified from wound chambers also were found to express mRNA for IL-1beta, IL-10, TNF-alpha, JE/MCP, MIP-1alpha, MIP-1beta, MIP-2, PDGF-A, TGF-beta1, and M-CSF. The addition of IL-1beta (1-100 ng/ml), a critical mediator in wound healing, to fibrocytes isolated from human peripheral blood induced the secretion of chemokines (MIP-1alpha, MIP-1beta, MCP-1, IL-8, and GRO alpha), hemopoietic growth factors (IL-6, IL-10, and macrophage-CSF), and the fibrogenic cytokine TNF-alpha. By contrast, IL-1beta decreased the constitutive secretion of type I collagen as measured by ELISA. Additional evidence for a role for fibrocytes in collagen production in vivo was obtained in studies of livers obtained from Schistosoma japonicum-infected mice. Mouse fibrocytes localized to areas of granuloma formation and connective matrix deposition. We conclude that fibrocytes are an important source of cytokines and type I collagen during both the inflammatory and the repair phase of the wound healing response. Furthermore, IL-1beta may act on fibrocytes to effect a phenotypic transition between a repair/remodeling and a proinflammatory mode.     

Monitoring glutamate release during LTP with glial transporter currents

Aminopeptidase (AP) A is a transmembrane type II molecule widely distributed in mammalian tissues. Since APA expression may be absent in renal cell carcinoma (RCC), it is possible that there is an altered regulation or other defect of APA upon malignant transformation of proximal tubular cells. However, investigations into the regulation of APA on tumour cells are rare. We report, for the first time, that both transforming growth factor-beta 1 (TGF-beta1) and tumour necrosis factor-alpha (TNF-alpha) down-regulate APA mRNA as well as protein expression in renal tubular epithelial cells and RCC cells in culture. In addition to this, both cytokines decrease dipeptidylpeptidase (DP) IV/CD26 mRNA, but not APN/CD13 mRNA expression. Otherwise, IL-4 and IL-13 increase CD13 as well as CD26 expression, but do not alter APA expression. Interferon-alpha (IFN-alpha), IFN-beta and IFN-gamma increase mRNA expression of all the three membrane ectopeptidases, whereas IL-1, IL-6, IL-7, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to be without any significant effect. Treatment of cultured cells with cAMP-increasing agents, such as 8-bromo-cAMP or A23187, results in an increase in APA and DPIV/CD26, but no change in APN/CD13 mRNA expression or even a decrease in it. Furthermore, AP inhibitors can influence APA mRNA expression, since bestatin causes an increase in APA expression in a time- and dose-dependent manner, whereas bestatin does not change CD13 or CD26 expression. No difference could be found with respect to the modulation by different mediators between RCC cells and renal epithelial cells, though permanent tumour cell lines such as Caki-1 and Caki-2 may have lost some of the normally expressed peptidases.     

Computer-based neuroanatomy laboratory for medical students

Bone is one of the most common sites of metastasis in melanoma and breast cancer cells. Human melanoma (A375M) and human breast cancer (MDA-MB-231) cells form osteolytic bone metastasis in vivo when these tumor cells are injected into the left ventricles of BALB/c nude mice. These tumor cells promote bone resorption in the in vitro neonatal murine calvaria organ culture system by indirectly stimulating the production of a bone resorption-inducing factor (or factors) from human osteoblast-like cells. This secreted factor was identified as interleukin-11 (IL-11). Although many cytokines and hormones were associated with IL-11 production from osteoblasts, transforming growth factor-beta (TGF-beta) was found to be involved in the promotion of IL-11 production from osteoblasts, because the addition of a neutralizing anti-TGF-beta antibody decreased the production of IL-11. However, these tumor cells did not produce TGF-beta by themselves. We found that they enhanced IL-11 production by activating latent TGF-beta produced from osteoblast-like cells. Our results indicate that metastatic tumor cells induce osteolysis by activating TGF-beta, which leads IL-11 production from osteoblasts to promote bone resorption.     

Inhibition of tissue factor surface expression in human peripheral blood monocytes exposed to cytokines

Interleukin (IL)-4, IL-10, IL-13 and transforming growth factor beta (TGF-beta) are known to regulate several monocyte functions, including inhibition of the synthesis of different cytokines. Using quantitative RT-PCR and flow cytometry analysis we investigated the effects of these cytokines on bacterial lipopolysaccharide (LPS)-induced tissue factor (TF) expression in human monocytes. The effects of IL-4 and IL-10 on monocyte chemoattractant protein-1 (MCP-1)-and C-reactive protein (CRP)-induced TF expression were also studied. A direct comparison revealed that IL-4, IL-10 and IL-13 all down-regulated LPS-induced TF expression in a concentration-dependent manner without the need for priming. In contrast, TGF-beta required 4 h of priming to inhibit TF expression induced by LPS. IL-10 was the most powerful inhibitor, causing almost complete inhibition at 5 ng/ml. IL-4 and IL-13 exhibited a significantly lower inhibitory capacity even at concentrations of 100 ng/ml. IL-4 and IL-10 showed similar concentration-dependent inhibition of MCP-1- and CRP-induced TF expression. We also showed that the regulatory effect of the interleukins occurred at the mRNA level. In vivo, these inhibitory cytokines may play an important regulatory role in preventing thrombosis. IL-10, in particular, may be a possible candidate as a TF-preventing drug.     

Differential modulation of B7-1 and B7-2 isoform expression on human monocytes by cytokines which influence the development of T helper cell phenotype

The co-stimulatory molecules B7-1/B7-2 expressed on the surface of antigen-presenting cells have been suggested to influence the development of T helper 1 (Th1)-versus Th2-immune responses. These studies were conducted to elucidate the effect of immunoregulatory cytokines which influence the development of Th1/Th2 immune responses on the expression of the B7 isoforms B7-1 and B7-2 on resting and activated human monocytes and B cells. Interleukin (IL)-4 and IL-10, which induce the development of Th2 immune responses, down-regulated B7-2 and moderately up-regulated B7-1 expression on resting CD14+ monocytes in peripheral blood mononuclear cells. Interferon-gamma (IFN-gamma), which induces the development of Th1 immune responses, enhanced the expression of both B7-1 and B7-2 isoforms. Tumor necrosis factor (TNF)-alpha, which elicits both Th1- and Th2 characteristics depending on experimental conditions, down-regulated B7-2 but did not alter B7-1 expression. The effect of TNF-alpha and B7-2 expression is not mediated through endogenously produced IL-10, as addition of anti-IL-10 antibodies did not restore B7-2 expression. None of the other cytokines tested, including IL-1 alpha, IL-1 beta, IL-2, IL-5, IL-6, IL-12, granulocyte/macrophage colony-stimulating factor (GM-CSF), and transforming growth factor (TGF)-alpha, modulated the expression of B7 isoforms on resting monocytes. Lipoolysaccharide stimulation of monocytes down-regulated B7-2 and up-regulated B7-1 expression in a manner similar to IL-10. The expression of B7-1 and B7-2 on purified B cells were not altered by any of the cytokines tested, including IL-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IFN-gamma, TNF-alpha, TGF-alpha and GM-CSF. Taken together, our results suggest that the cytokines which induce Th1/Th2 immune responses exert differential effects on B7 isoform expression on resting monocytes but have no effect on resting or activated B cells.     

Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.     

Tissue distribution of DNA adducts in rats treated by intramammillary injection with dibenzo[a,l]pyrene, 7,12-dimethylbenz[a]anthracene and benzo[a]pyrene

Recombinant human bone morphogenetic protein (rhBMP-2) was examined for its in vitro effects on biochemical markers representing osteoblast phenotype. Primary cultures of fetal rat calvarial osteoblasts were used in this study. The results indicated that rhBMP-2 stimulated alkaline phosphatase activity, parathyroid hormone (PTH)-induced cyclic AMP production, and collagen biosynthesis in a dose-dependent manner in confluent cultures. The percent collagen synthesis also increased in a dose-dependent manner. Alkaline phosphatase activity was stimulated in a time-dependent manner by rhBMP-2 that reached its maximum 5 days after initiation. Cycloheximide (2 micrograms/ml) inhibited rhBMP-2-stimulated alkaline phosphatase indicating de novo protein synthesis of the enzyme. Transforming growth factor-beta 1 (TGF-beta 1)-induced inhibition of alkaline phosphatase activity observed in confluent primary cultures was completely abolished by rhBMP-2 at a concentration that was 43 times greater than the TGF-beta 1 concentration. Also, rhBMP-2 produced a small stimulation of alkaline phosphatase activity in cells grown in the absence of ascorbic acid; however, the effect was greatly enhanced in cells cultivated in the presence of ascorbic acid (50 micrograms/ml). In view of the potentiating effect of ascorbic acid on rhBMP-2-induced stimulation of alkaline phosphatase, we speculate that ascorbic acid could amplify the osteoinductive effects of rhBMP-2 and thereby augment the efficacy of the BMP when used as bone repair material in vivo. rhBMP-2 (4.3-86 ng/ml) did not exhibit mitogenic effects on cultured osteoblasts. These data suggest that rhBMP-2 has the ability to induce expression of various markers associated with the osteoblast phenotype in primary cultures of fetal rat calvarial osteoblasts. In addition, we speculate that TGF-beta 1 may play a regulatory role in BMP-induced bone formation and ascorbic acid may potentiate the effects of rhBMP-2 in vivo.     

Tyrosine kinase inhibitor herbimycin A reduces the stability of cyclin-dependent kinase Cdk6 protein in T-cells

The induction of macrophage-deactivating (interleukin-10 [IL-10] and transforming growth factor beta [TGF-beta] and macrophage-activating (IL-1, IL-6, and tumor necrosis factor alpha [TNF-alpha] cytokines by lipoarabinomannan (LAM) from pathogenic Mycobacterium tuberculosis Erdman and H37Rv strains (ManLAM) and nonpathogenic mycobacteria (AraLAM) in human blood monocytes was examined. ManLAM was significantly less potent in induction of TNF-alpha, IL-1, IL-6, and IL-10 protein and mRNA, whereas its ability to induce TGF-beta was similar to that of AraLAM. Differences in induction of TNF-alpha mRNA by the two LAM preparations only became apparent at late time points of culture (24 h). The induction of TNF-alpha and IL-1 by purified protein derivative of M. tuberculosis was significantly stronger than that by ManLAM. Pretreatment of monocytes with ManLAM did not, however, interfere with cytokine induction by lipopolysaccharide or AraLAM. The extensive mannosyl capping of arabinose termini of ManLAM may underlie the lack of ability to induce some cytokines (IL-1, TNF-alpha, and IL-10) and the retained ability to induce TGF-beta. The latter may have a role in shifting the cytokine milieu in favor of survival of M. tuberculosis.