Enhancing effect of IL-1, IL-17, and TNF-alpha on macrophage inflammatory protein-3alpha production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines.

Macrophage inflammatory protein (MIP)-3alpha is a chemokine involved in the migration of T cells and immature dendritic cells. To study the contribution of proinflammatory cytokines and chemokines to the recruitment of these cells in rheumatoid arthritis (RA) synovium, we looked at the effects of the monocyte-derived cytokines IL-1beta and TNF-alpha and the T cell-derived cytokine IL-17 on MIP-3alpha production by RA synoviocytes. Addition of IL-1beta, IL-17, and TNF-alpha induced MIP-3alpha production in a dose-dependent manner. At optimal concentrations, IL-1beta (100 pg/ml) was much more potent than IL-17 (100 ng/ml) and TNF-alpha (100 ng/ml). When combined at lower concentrations, a synergistic effect was observed. Conversely, the anti-inflammatory cytokines IL-4 and IL-13 inhibited MIP-3alpha production by activated synoviocytes, but IL-10 had no effect. Synovium explants produced higher levels of MIP-3alpha in RA than osteoarthritis synovium. MIP-3alpha-producing cells were located in the lining layer and perivascular infiltrates in close association with CD1a immature dendritic cells. Addition of exogenous IL-17 or IL-1beta to synovium explants increased MIP-3alpha production. Conversely, specific soluble receptors for IL-1beta, IL-17, and TNF-alpha inhibited MIP-3alpha production to various degrees, but 95% inhibition was obtained only when the three receptors were combined. Similar optimal inhibition was also obtained with IL-4, but IL-13 and IL-10 were less active. These findings indicate that interactions between monocyte and Th1 cell-derived cytokines contribute to the recruitment of T cells and dendritic cells by enhancing the production of MIP-3alpha by synoviocytes. The inhibitory effect observed with cytokine-specific inhibitors and Th2 cytokines may have therapeutic applications.     

Molecular inhibition of histone deacetylation results in major enhancement of the production of IL-1beta in response to LPS

It has been postulated that the progression of human pregnancy to term is, in part, the result of a relative maternal Th(2) immunological state. This can be activated in some cell types by modifying DNA methylation and histone acetylation status. We demonstrate that the molecular inhibition of histone deacetylation, using trichostatin A (TSA), in human choriodecidual explants leads to a massive increase in lipopolysaccharide (LPS)-stimulated IL-1beta. The inhibition of histone deacetylation had no effect on LPS-stimulated TNF-alpha production or production of the other cytokines studied (IL-10, IL-1 receptor antagonist). The molecular inhibition of DNA methylation and histone deacetylation, using 5-aza-2'-deoxycytidine and TSA, respectively, in human choriodecidual explants also results in an increase in the basal production of TNF-alpha but not that of IL-1beta. The differential response is unique, and the relative uncoupling of IL-1beta and TNF-alpha responsiveness may have importance in other biological systems and provide new therapeutic targets for pathologies where upregulation of IL-1beta is known to be a causative factor.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood: II. Application to rheumatoid arthritis and osteoarthritis.

Rheumatoid arthritis (RA) is an immune disease in which the pathological immune reaction is thought to be initiated by the presentation of an (auto) antigen or superantigen by MHC class II positive cells to CD4 T cells. These successive immunological events can be studied by the cytokines produced at the different stages. Cytokine secretion by stimulated cells in autologous diluted whole blood has allowed the study of the immune profile characteristic of rheumatoid arthritis. The pattern of RA patient whole blood cells cultured in autologous blood is characterized by hyperactivity of the mononuclear cells with high secretion of IL-1 beta, TNF-alpha and IL-6 and low production of IFN-gamma, in comparison with the normal (N) and osteoarthrosis (OA) populations. The IL-2 secretion pattern is unique, arising from production followed by consumption. This production-consumption turnover is the most elevated in the RA group. The T cells are indeed activated in rheumatoid arthritis but regulatory events suppress some of their functions. A correlation was found between the inflammatory proteins and mediators of cellular immunity and macrophagic function: IL-1 beta and the sedimentation rate; IL-6 and fibrinogen; TNF-alpha and the number of blood monocytes. The secretion of OA-stimulated whole blood cells was similar to RA for two monokines (overproduction of TNF-alpha and IL-6) and different for IL-1 beta, not different from normal in OA. Stimulated whole blood cell cytokine secretion profile from RA and OA groups, was the same as previously observed in synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ethanol induces the production of cytokines via the Ca2+, MAP kinase, HIF-1alpha, and NF-kappaB pathway

In the present study, we sought to investigate the signal transduction pathways of expression of cytokines in the ethanol-stimulated human mast cell line, HMC-1. Ethanol significantly increased the intracellular calcium level in HMC-1. Ethanol also significantly enhanced IL-6, TNF-alpha, and TGF-beta1 production compared with media control, but did not significantly affect the IL-1beta production. After 8 h of stimulation, ethanol increased mRNA and protein expression levels of TNF-alpha and TGF-beta1 in HMC-1. The increased cytokine level was significantly inhibited by BAPTA-AM, PD98059, and SB203580. These inhibitors also inhibited ethanol-induced ERK and p38 MAPK phosphorylation. Ethanol resulted in a great increase in protein levels and promoter activity driving luciferase expression of HIF-1alpha and NF-kappaB in HMC-1 cells, but it did not affect on HIF-1alpha mRNA expression. Our observations show that calcium, MAPK activation, HIF-1alpha, and NF-kappaB are necessary for ethanol-induced TNF-alpha and TGF-beta1 expression. These results may have important implications for the study of alcohol-related diseases.     

Cell density regulates neutrophil IL-8 synthesis: role of IL-1 receptor antagonist and soluble TNF receptors

Although cytokine synthesis in polymorphonuclear leukocytes (PMN) was shown to be modulated by soluble mediators, the impact of microenvironmental conditions has not been elucidated. In this study, we investigated the effect of cell density on cytokine release from human neutrophils. PMN were cultured at various cell densities (10 x 10(6) PMN/ml; 60 x 10(6) PMN/ml), and LPS-induced release of cytokines was quantified by ELISA technique. Upon an increase in PMN density, secretion of the CXC chemokine IL-8 was progressively reduced. This effect was paralleled by a decrease in IL-8 mRNA. In contrast, TNF-alpha and IL-1beta rose proportionally with increasing cell density. The inhibition of IL-8 secretion was reproduced by conditioned media of PMN at high cell density, but was not affected by blocking beta(2) integrin-dependent adhesion. When analyzing the supernatant of LPS-challenged neutrophils, large amounts of soluble TNFRs p55 and p75 (sTNFRI, sTNFRII), and IL-1R antagonist (IL-1RA), rising constantly with the cell density, were detected. Interestingly, combined blocking of the bioactivities of these mediators completely restored neutrophil IL-8 secretion at high cell densities, with the anti-IL-1RA Ab being the more potent agent. Moreover, combined application of exogenous IL-1RA and sTNFRs to 10 x 10(6) PMN/ml reproduced the suppression of IL-8 generation. We conclude that neutrophil IL-8 synthesis is autoregulated, being suppressed under conditions of high cell density. IL-1RA and sTNFRs, accumulating under these circumstances, seem to be centrally involved in this regulatory mechanism by interfering with the IL-1beta- and TNF-alpha-dependent IL-8 generation. This feedback mechanism may control further neutrophil recruitment and activation in a neutrophil-rich environment, thereby preventing tissue destruction.     

Synovial tissue macrophage as a source of the chemotactic cytokine IL-8

Cells of the synovial microenvironment may recruit neutrophils (PMN) and lymphocytes into synovial fluid, as well as lymphocytes into the synovial tissues, of arthritic patients. We have investigated the production of the chemotactic cytokine IL-8 by using sera, synovial fluid, synovial tissue, and macrophages and fibroblasts isolated from synovial tissues from 75 arthritic patients. IL-8 levels were higher in synovial fluid from rheumatoid (RA) patients (mean +/- SE, 14.37 +/- 5.8 ng/ml), compared with synovial fluid from osteoarthritis patients (0.135 +/- 17 ng/ml) (p less than 0.05) or from patients with other arthritides (5.52 +/- 5.11 ng/ml). IL-8 from RA sera was 8.44 +/- 2.33 ng/ml, compared with nondetectable levels found in normal sera. IL-8 levels from RA sera and synovial fluid were strongly positively correlated (r = 0.96, p less than 0.05). Moreover, RA synovial fluid chemotactic activity for PMN in these fluids was inhibited 40 +/- 5% upon incubation with neutralizing polyclonal antibody to IL-8. Synovial tissue fibroblasts released only small amounts of constitutive IL-8 but could be induced to produce IL-8 by stimulation with either IL-1 beta, TNF-alpha, or LPS. In contrast, unlike normal PBMC or alveolar macrophages, macrophages isolated from RA synovial tissue constitutively expressed both IL-8 mRNA and antigenic IL-8. RA synovial macrophage IL-8 expression was not augmented by incubation with either LPS, TNF-alpha, or IL-1 beta. Immunohistochemical analysis of synovial tissue showed that a greater percentage of RA macrophages than osteoarthritis macrophages reacted with anti-IL-8. Whereas macrophages were the predominant cell for immunolocalization of IL-8, less than 5% of synovial tissue fibroblasts were positive for immunolocalized IL-8. These results suggest that macrophage-derived IL-8 may play an important role in the recruitment of PMN in synovial inflammation associated with RA.     

Differential regulation of TNF-alpha and IL-1beta production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors

The effect of selective PDE-I (vinpocetine), PDE-III (milrinone, CI-930), PDE-IV (rolipram, nitroquazone), and PDE-V (zaprinast) isozyme inhibitors on TNF-alpha and IL-1beta production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-alpha production, but only partially inhibited IL-1beta at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-alpha, but had no effect on IL-1beta production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-alpha and IL-1beta production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man.     

Recombinant human IL-1 beta and TNF-alpha stimulate production of IL-1 alpha and IL-1 beta by vascular smooth muscle cells and IL-1 alpha by vascular endothelial cells

Vascular endothelial cells (EC) produced IL-1 alpha but not IL-1 beta into extracellular fluids. Vascular smooth muscle cells (SMC), on the other hand, produced both IL-1 alpha and IL-1 beta, and IL-1 beta produced was much higher than IL-1 alpha. The addition of recombinant human IL-1 beta or recombinant human TNF-alpha significantly enhanced IL-1 alpha production in EC, and IL-1 alpha and IL-1 beta production in SMC. IL-1 beta release was not observed even when EC were stimulated with TNF-alpha. These results suggest that the species of released form of IL-1 are different in different cell types and that cytokines enhance IL-1 alpha and IL-1 beta production in SMC and IL-1 alpha production in EC.     

Macrophage inflammatory protein-3 beta enhances IL-10 production by activated human peripheral blood monocytes and T cells.

We report that the addition of human macrophage inflammatory protein-3 beta (MIP-3 beta) to cultures of human PBMCs that have been activated with LPS or PHA results in a significant enhancement of IL-10 production. This effect was concentration-dependent, with optimal MIP-3 beta concentrations inducing more than a 5-fold induction of IL-10 from LPS-stimulated PBMCs and a 2- to 3-fold induction of IL-10 from PHA-stimulated PBMCs. In contrast, no significant effect on IL-10 production was observed when 6Ckine, the other reported ligand for human CCR7, or other CC chemokines such as monocyte chemoattractant protein-1, RANTES, MIP-1 alpha, and MIP-1 beta were added to LPS- or PHA-stimulated PBMCs. Similar results were observed using activated purified human peripheral blood monocytes or T cells. Addition of MIP-3 beta to nonactivated PBMCs had no effect on cytokine production. Enhancement of IL-10 production by MIP-3beta correlated with the inhibition of IL-12 p40 and TNF-alpha production by monocytes and with the impairment of IFN-gamma production by T cells, which was reversed by addition of anti-IL-10 Abs to the cultures. The ability of MIP-3 beta to augment IL-10 production correlated with CCR7 mRNA expression and stimulation of intracellular calcium mobilization in both monocytes and T cells. These data indicate that MIP-3 beta acts directly on human monocytes and T cells and suggest that this chemokine is unique among ligands binding to CC receptors due to its ability to modulate inflammatory activity via the enhanced production of the anti-inflammatory cytokine IL-10.     

Production of IL-10, TNF-alpha, IFN-gamma, TGF-beta1 by different populations of erythroid cells derived from human embryonal liver

It has previously been determined that erythroid cells of mice are capable of expressing such cytokines as interleukin (IL) 1 alpha and beta, IL-4, IL-6, interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta). It has been shown that glycophorin A(+) (GlA(+)) and antigen erythroblasts (AG-EB(+)) (both human erythroid cells of embryonic origin) are also capable of producing a series of cytokines such as IL-1 beta, IL-2, IL-4 and IL-6. The aim of this work was to study the capacity of erythroid cells from human embryonic liver to produce such cytokines as IFN-gamma, TGF-beta1, tumour necrosis factor alpha (TNF-alpha) and IL-10. The erythroid cells were isolated by means of antibodies specific to erythroblasts (GlA and AG-EB), as well as those from single erythroid colonies. The production level of some cytokines varies insignificantly under the action of erythropoietin (Epo) and quantitatively differs in GlA(+) and AG-EB(+) erythroid cells. Hence, the erythroid cells express IFN-gamma, TGF-beta1, TNF-alpha and IL-10. The erythroid cells could be involved through the production of these cytokines in the regulation of such processes as self-renewal, proliferation and differentiation of cells of other blood-forming sites.     

Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.     

Comparative effects of tumor necrosis factor-alpha and IL-1 beta on mitogen-induced T cell activation

The effect of rTNF-alpha on human T cell function was examined and compared with that of rIL-1 beta by assessing the ability of each cytokine to support mitogen-induced proliferation, IL-2 production, and IL-2R expression. TNF-alpha and IL-1 beta each enhanced DNA synthesis induced by PHA or immobilized mAb to the CD3 molecular complex. In addition, each cytokine increased the number of cells entering the G1 phase of the cell cycle and augmented IL-2R expression. The combination of optimal concentrations of these factors supported these responses to a greater extent than either cytokine alone, suggesting that T cell responsiveness is independently regulated by the action of at least two separate monocyte derived cytokines. Whereas TNF-alpha had little effect, IL-1 beta augmented IL-2 mRNA expression and IL-2 production by mitogen-stimulated cells. Furthermore, IL-1 beta enhanced proliferation with increasing length of culture. Whereas TNF-alpha also enhanced proliferation late in culture, it was less effective in this regard than IL-1 beta. Thus, IL-1 beta and TNF-alpha augment mitogen-induced T cell proliferation by increasing the number of cells initially activated and by promoting subsequent cell cycle progression. They differ, however, in their capacity to promote IL-2 mRNA and IL-2 production and therefore ongoing T cell proliferation.     

Reduction of inflammatory cytokines and prostaglandin E2 by IL-13 gene therapy in rheumatoid arthritis synovium

The rheumatoid arthritis (RA) joint is characterized by an inflammatory synovial pannus which mediates tissue destruction. IL-13 is a cytokine that inhibits activated monocytes/macrophages from secreting a variety of proinflammatory molecules. The aim of this study was to examine whether gene therapy-delivered IL-13 could reduce the production of key proinflammatory mediators in RA synovial tissue (ST) explants. Adenoviral vectors encoding the genes for human IL-13 (AxCAIL-13) and bacterial beta-galactosidase were generated and examined for protein production. Vectors were used to infect RA ST explants and RA synovial fibroblasts, and conditioned medium (CM) was collected at various times for analysis by ELISA and competitive immunoassay. AxCAIL-13 decreased the production of RA ST explant proinflammatory IL-1beta by 85% after 24 h. Likewise, TNF-alpha levels were decreased by 82 and 75% whereas IL-8 levels were reduced 54 and 82% after 24 and 48 h, respectively, in RA ST explant CM. Monocyte chemotactic protein-1 concentrations were decreased by 88% after 72 h in RA ST explant CM. RA ST explant epithelial neutrophil-activating peptide-78 concentrations were decreased 85 and 94% whereas growth-related gene product-alpha levels were decreased by 77 and 85% at 24 and 48 h, respectively, by AxCAIL-13. Further, IL-13 significantly decreased PGE2 and macrophage inflammatory protein-1alpha production. These results demonstrate that increased expression of IL-13 via gene therapy may decrease RA-associated inflammation by reducing secretion of proinflammatory cytokines and PGE2.