Lipopolysaccharide-Enhanced Expression of Interleukin-6 in Dibutyryl Cyclic AMP-Differentiated Rat C6 Glioma

Abstract: Rat C6 glioma synthesizes a low basal level of interleukin-6 (IL-6). Stimulation with 10 µg/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 m M N ⁶, O ^2'-dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP-differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL-6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 µg/ml of LPS and 1 m M dbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL-6 mRNA concentration and IL-6 immunoreactivity, whereas titration of the biological activity indicated a 100-fold increase in the secreted IL-6 activity. The enhanced secretion of IL-6 is correlated with the induction of differentiation. Chromatography on heparin-Sepharose and on DEAE-5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL-6-like activities could be neutralized by an anti-murine IL-6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending on their degree of differentiation.     

In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumor-bearing mice

We investigated the capacity of several recombinant cytokines to induce IL-6 in vivo in both normal and tumor-bearing (TB) mice. Intravenous administration of human rhTNF-alpha, rhIL-1, rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma were all capable of inducing circulating IL-6. rhTNF-alpha administration caused the greatest induction of IL-6. TB animals consistently produced more IL-6 in response to rhTNF-alpha than did normal mice (2 h after 4 micrograms rhTNF-alpha, TB = 24,100 HGF U/ml, non-TB = 3600 HGF U/ml of IL-6). A single daily i.v. dose of rhTNF-alpha (4 micrograms/mouse/day) for 5 days led to decreased IL-6 induction in TB animals by day 3 of treatment (peak levels of IL-6, day 1 = 72,800 HGF U/ml, day 3 = 23,400 HGF U/ml, day 5 = 26,400 HGF U/ml). rhIL-1 administration also resulted in considerable IL-6 production, although peak values were less than those resulting from administration of rhTNF-alpha. Administration of rhIL-1 induced similar IL-6 levels (TB = 10,025 and non-TB = 10,600 HGF U/ml) in TB and normal mice. Single high doses of rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma induced lower but consistent levels of circulating IL-6 in mice with and without tumor. In addition, the sera of untreated TB mice contained levels of IL-6 which paralleled the extent of tumor burden (serum IL-6 in day 30 MCA 106 TB mice = 420 HGF U/ml). The detection of de novo IL-6 was also confirmed in animals bearing tumors of different histologies (the MCA 102 sarcoma, MCA 38 adenocarcinoma, and B16 melanoma). At no time was IL-6 measurable in the sera of untreated normal mice. The identification of IL-6 was verified by neutralization studies using specific antimurine IL-6 antibody. Although the exact role of IL-6 in TB animals remains to be elucidated, its known pleotrophic immune and metabolic effects may be important in the host response to malignancy.     

Expression of cytokine mRNA and protein in joints and lymphoid organs during the course of rat antigen-induced arthritis

Cytokine expression was assessed during antigen-induced arthritis (AIA) in synovial membrane (SM), inguinal lymph node (LN), and spleen using competitive RT-PCR and sandwich ELISA. In the SM, early elevations of IL-1β and IL-6 mRNA (by 6 hours; 450- and 200-fold, respectively) correlated with the joint swelling; a 6-fold increase in tumor necrosis factor α (TNFα) was not significant. Not only IL-2 and IFN-γ (which increased 10,000-fold and 200-fold, respectively), but also IL-5 and IL-10, increased acutely (6 hours – day 1; 3-fold and 35-fold, respectively) in the SM. In general, the protein levels in the SM for IL-1β, IL-6, TNFα, IFN-γ, IL-4, and IL-10 (increase from 4-fold to 15-fold) matched the course of mRNA expression. In the inguinal LN, there were early mRNA elevations of IL-6 (a 2.5-fold increase by 6 hours, which correlated positively with the joint swelling) and IL-2 (4-fold by 6 hours), as well as later rises of IL-4 and IL-5 (2.5- and 4-fold, respectively, by day 3). No significant elevations of the corresponding proteins in this tissue were observed, except for IL-1β (by day 6) and IL-10 (by day 1). In the spleen, there were significant mRNA elevations at 6 hours of IL-1β (1.5-fold), IL-6 (4-fold; positively correlated with the joint swelling), IFN-γ (3-fold), and IL-2 (7- to 10-fold). IL-5 and IL-10 (2- and 3-fold, respectively) peaked from 6 hours to day 3 in the spleen. Increases of the corresponding proteins were significant in comparison with day 0 only in the case of IL-2 (day 6). By day 6 (transition to the chronic phase), the mRNA for cytokines declined to or below prearthritis levels in all the tissues studied except for IL-1β in the SM and IL-6 in the spleen. AIA is thus characterized by four phenomena: early synovial activation of macrophages, T helper (Th)1-like, and Th2-like cells; late, well-segregated Th2-like responses in the inguinal LN; late, overlapping Th1-like/Th2-like peaks in the spleen; and chronic elevation of synovial IL-1β mRNA and spleen IL-6 mRNA.     

IL-1 and tumor necrosis factor. Similarities and differences in stimulation of expression of alternative pathway of complement and IFN-beta 2/IL-6 genes in human fibroblasts

IL-1 and TNF induced concentration-related increases in the synthesis of factor B, C3, and IFN-beta 2/IL-6 in human skin fibroblasts. Effects of both stimuli were apparent with concentrations as low as 0.1 ng/ml and maximal responses were observed between 1 and 10 ng/ml; only for IL-1 induction of IFN-beta 2/IL-6 was there a further increase in response up to 100 ng/ml. For factor B and C3, maximal increases induced by IL-1 and TNF were similar: 119- and 109-fold for factor B and 15-fold and 11-fold for C3, respectively. Although both IL-1 and TNF increase synthesis of factor B and C3 in hepatocytes, the increases observed in fibroblasts were approximately 50- and 8-fold more for factor B and C3, respectively. Neither protein synthesis nor mRNA for IFN-beta 2/IL-6 was present in HepG2 cells either before or after stimulation with IL-1 or TNF. In contrast to the similarities between the effects of IL-1 and TNF on synthesis of factor B, C3, and IFN-beta 2/IL-6, only TNF increased synthesis of factor H. Because TNF induces membrane IL-1 in fibroblasts, it is possible to speculate that the effects of TNF on fibroblasts are due to induction of IL-1. An autocrine action of TNF through IL-1 is possible for TNF-induced synthesis of IFN-beta 2/IL-6, but the effects of TNF on synthesis of factor B, C3, and factor H indicated that TNF has effects on fibroblasts separate from IL-1. The effects of IL-1 and TNF on the synthesis of factor B and C3 in fibroblasts may be a part of an acute phase response occurring at a local level. However, the large responses in synthesis of factor B and C3 to IL-1 and TNF may suggest that factor B and C3 have a role, as yet undescribed, in tissues in addition to the role these proteins are known to play in inflammation.     

IL-2 and IL-6 synergize to augment the pore-forming protein gene expression and cytotoxic potential of human peripheral blood T cells

Our previous studies have demonstrated that high dose IL-2 (1000 U/ml) alone can induce human peripheral blood T cell pore-forming protein (PFP) mRNA expression and cytotoxic potential. We now report that the levels of IL-2 needed to induce these effects in T cells can be significantly reduced in the presence of IL-6. IL-6 and suboptimal doses of IL-2 (10 U/ml) were found to costimulate PFP mRNA expression and cytotoxic potential in resting human peripheral blood T cells, whereas IL-6 or low dose IL-2 alone had no effect. The induction of T cell PFP mRNA by IL-2/IL-6 was extremely rapid and increases in both PFP mRNA expression and cytotoxic potential were IL-6 dose dependent. The costimulatory effect of IL-6 did not appear to involve the IL-2/IL-2R pathway in as much as IL-6 did not induce IL-2 production or detectably increase IL-2R surface expression in T cells. These findings, in addition to the rapid induction of PFP mRNA by IL-2/IL-6, suggested that IL-6 can directly and independently provide an additional signal to augment the differentiation of CTL. In contrast to the results observed in T cells, IL-6 and IL-2 could enhance CD3- large granular lymphocyte (LGL) NK activity, but IL-6 either alone or in combination with IL-2 had no effect on constitutive PFP mRNA expression in resting LGL. These data further confirm that different mechanisms may be responsible for lymphokine activation of CTL and LGL in human peripheral blood. In particular it appears that IL-6 acts as a costimulatory signal with IL-2 in generating CTL and that IL-6 functions in part by acting in synergy with IL-2 to induce PFP, a major lytic protein involved in lymphocyte cytotoxicity.     

Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts.

Hepatocyte growth factor (HGF) functions as a hepatotrophic and renotrophic factor for regeneration of the liver and kidney. When 1 ng/ml of interleukin-1 alpha (IL-1 alpha) or interleukin-1 beta (IL-1 beta) was added to cultures of human skin fibroblasts, the production of HGF was 5-6 fold higher than levels in the controls. HGF mRNA level in the cells was increased to 4-fold higher levels at 6 h after exposure to IL-1 alpha. Tumor necrosis factor-alpha and interferon-gamma but no other cytokine tested had slightly stimulatory effects on HGF production. The tumor promoter, tetradecanoylphorbol 13-acetate (TPA) markedly enhanced the stimulatory effect of IL-1 alpha and IL-1 beta on the production of HGF. The stimulatory effect of both IL-1 alpha and IL-1 beta and the synergistical stimulation with TPA were completely abrogated by 10 ng/ml TGF-beta 1 or 1 microM dexamethasone. These results suggest that IL-1 alpha and IL-1 beta are positive regulators for expression of the HGF gene and are likely have a role in regeneration of tissues following the occurrence of inflammatory diseases.     

Decreased steady-state insulin-like growth factor binding protein-3 (IGFBP-3) mRNA level is associated with differentiation of cultured porcine myogenic cells

Insulin-like growth factor binding proteins (IGFBPs) affect the biological activity of IGF-I in several cell types, including cultured muscle cells. Additionally, at least one of the IGFBPs, IGFBP-3, has been shown to have IGF-independent effects on cell proliferation. Numerous studies have shown that immortalized muscle cell lines produce various IGFBPs, but to date no muscle cell line has been reported to produce IGFBP-3 protein or mRNA. Unlike muscle cell lines, primary cultures of porcine embryonic myogenic cells express IGFBP-3 mRNA and secrete a protein that is immunologically identifiable as IGFBP-3. Additionally, steady-state IGFBP-3 levels change significantly during differentiation. Here we report that differentiation of porcine myogenic cells in an IGFBP-3-free medium is accompanied by reduced steady-state IGFBP-3 mRNA levels. Steady-state levels of IGFBP-3 mRNA decreased approximately sevenfold (P < .05) during differentiation and then increased to predifferentiation levels once differentiation was complete. Addition of TGF-beta1 (0.5 ng/ml) to porcine myogenic cultures suppressed fusion and resulted in a sevenfold increase in steady-state IGFBP-3 mRNA and a 1.8-fold increase in IGFBP-3 protein levels as compared to untreated control cultures (P < .05). Results suggest that alterations in IGFBP-3 mRNA and protein may play a role in differentiation of porcine embryonic muscle cells.     

IL-2 induces expression of serine protease enzymes and genes in natural killer and nonspecific T killer cells

The expression of serine protease genes was examined in murine NK cells that were purified by panning spleen cells with PMA. Although unstimulated NK cells were cytolytic, they were found not to express the C11 (chymotrypsin-like) mRNA. Culturing these cells in IL-2 (500 to 800 U/ml) for 5 to 7 days induced both the lytic activities and the protease enzymes by 20- to 30-fold. Concomitant to these activation events, the total steady state mRNA of both C11 and HF (trypsin-like) genes were also elevated. The activation of lysis, serine protease enzymes, and C11 and HF mRNA all peaked around day 5 in culture and was dose dependent. In order to exclude the possibility that PMA synergizes with IL-2 in this system, spleen cells from SCID mice, which contained mainly NK cells, were cultured under the same conditions (800 U/ml IL-2, with or without PMA) and PMA did not appear to enhance the expression of these mRNA. Similarly, IL-2 also induced the lytic activities, enzyme levels, and mRNA in the non-Ag-specific T killer cells isolated from spleens of normal mice. Lytic activity of T killer cells was not as high as the NK cells, however, the addition of PHA into the lytic assay resulted in enhanced lysis comparable to that of NK cells. These results showed that lytic activity increased along with protease enzyme levels and mRNA expression in both NK and resting T cells. Therefore, elevated levels of the protease enzymes could be one mechanism involved in optimal lytic activity of IL-2-induced lymphokine activated killer cells.     

Effects of cytokines on the production of lipoprotein lipase in cultured human macrophages

Macrophages are important cells in the pathogenesis of atherosclerosis because of their tendency to accumulate lipid and become transformed into foam cells. Cultured human monocyte-derived macrophages spontaneously secrete lipoprotein lipase (LPL), and LPL has been linked to increased lipid uptake by these cells. Because secretion of various macrophage products depends on activation by lymphokines, we studied the effects of immunoregulatory lymphokines on LPL secretion by cultured human macrophages. After culturing cells in RPMI 1640 medium with 20% fetal calf serum, recombinant human gamma-interferon (gamma-INF), interleukin-1 (IL-1), and interleukin-2 (IL-2) were added to the medium and LPL secretion was assessed by measuring LPL activity and/or LPL mass in the medium. Gamma-INF suppressed LPL production both when added to freshly plated cultures of human blood monocytes, as well as when added to monocyte/macrophages from mature cultures (day 6) that were producing large amounts of LPL. IL-1 inhibited medium LPL when added to freshly plated cultures, but not when added to mature cultures. On the other hand, IL-2 did not inhibit LPL in freshly plated cultures, but produced a dose-dependent suppression of LPL from mature cultures. None of the cytokines were cytotoxic to macrophages, and cells that were cultured in gamma-INF demonstrated partial recovery from LPL-suppressive doses of the cytokine. After exposure of cells to 50 U/ml of gamma-INF and 50 U/ml of IL-2 for 3 days, LPL mRNA levels, when expressed as LPL/gamma-actin ratios, were 42% and 53% of controls, respectively. Thus, activation of human macrophages in vitro by gamma-INF resulted in a suppression of LPL production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mice lacking serum amyloid P component do not necessarily develop severe autoimmune disease

Interleukin-10 (IL-10) is a cytokine with many regulatory functions. In particular, IL-10 exerts neutralizing effect on other cytokines, and therefore IL-10 is thought to have important therapeutic implications. Recent reports suggest that IL-10 regulates not only immunocytes but also collagen and collagenase gene expression in fibroblasts. In this study, we investigated the effect of IL-10 on gene expression of extracellular matrix (ECM) proteins, such as type I collagen, fibronectin, and decorin, in human skin fibroblasts. Results of Northern blot analysis showed that both collagen I and fibronectin mRNAs were downregulated, while decorin gene expression was enhanced by IL-10 (10 ng/ml) time-dependently (6-24 h). alpha1(I) collagen and fibronectin mRNAs were decreased to one-third and one-fourth, respectively, by 50 ng/ml IL-10, whereas decorin mRNA was increased up to 2.7-fold by 50 ng/ml IL-10. Response to IL-10 by scleroderma fibroblasts was similar to that in normal dermal fibroblasts, with decreased expression levels of collagen and fibronectin and induced decorin mRNA levels. Transforming growth factor-beta (TGF-beta) is a crucial fibrogenic cytokine which upregulates the mRNA expression of collagen and fibronectin, whereas it downregulates decorin mRNA expression in fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) has recently been shown to upregulate the type I collagen mRNA expression in cultured fibroblasts. We therefore examined whether IL-10 alters gene expression of ECM elicited by TGF-beta and MCP-1. Our results demonstrated that IL-10 downregulated the TGF-beta-elicited increase of mRNA expression of type I collagen and fibronectin, while partially recovering TGF-beta-elicited decrease of decorin expression in normal skin fibroblasts. By contrast, IL-10 did not alter the MCP-1-elicited upregulation of mRNA expression of either alpha1(I) collagen and decorin. Our data indicate that IL-10 differentially regulates TGF-beta and MCP-1 in the modulation of ECM proteins and therefore suggest that IL-10 plays a role in the regulation of tissue remodeling.     

IL-1 beta induces IL-6 expression in human orbital fibroblasts: identification of an anatomic-site specific phenotypic attribute relevant to thyroid-associated ophthalmopathy

Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we report that these fibroblasts, when treated with IL-1beta, express high levels of IL-6, a cytokine involved in B cell activation and the regulation of adipocyte metabolism. The magnitude of this induction is considerably greater than that in dermal fibroblasts and involves up-regulation of IL-6 mRNA levels. IL-1beta activates both p38 and ERK 1/2 components of the MAPK pathways. Disrupting these could attenuate the IL-6 induction. The up-regulation involves enhanced IL-6 gene promoter activity and retardation of IL-6 mRNA decay by IL-1beta. Dexamethasone completely blocked the effect of IL-1beta on IL-6 expression. Orbital fibroblasts also express higher levels of IL-6R than do skin-derived cells. When treated with rIL-6 (10 ng/ml), STAT3 is transiently phosphorylated. Thus, the exaggerated capacity of orbital fibroblasts to express high levels of both IL-6 and its receptor in an anatomic site-selective manner could represent an important basis for immune responses localized to the orbit in Graves' disease.     

Differentiation-dependent expression of obese (ob) gene by preadipocytes and adipocytes in primary cultures of porcine stromal-vascular cells

The relationship between obese (ob) gene expression and preadipocyte differentiation was examined in primary cultures of porcine stromal-vascular (S-V) cells by Northern-blot analysis using a pig ob cDNA probe. Isolated adipocytes expressed high levels of ob gene, but S-V cells did not express the ob gene. Cultures were seeded with fetal bovine serum (FBS) plus dexamethasone (Dex) for 3 days followed by ITS (insulin 5 μg/ml, transferrin 5 μg/ml, and selenium 5 ng/ml) treatment for 6 days. Detectable levels of ob mRNA first appeared at day 1 with very low activity of glycerol phosphate dehydrogenase (GPDH). Levels of ob mRNA increased in parallel with preadipocyte number or GPDH activity at the later times in cultures. The depletion of preadipocytes by complement-mediated cytotoxicity at day 3 of culture resulted in markedly decreased ob mRNA expression. Immunocytochemical analysis showed that ob protein was localized in the cytosol of preadipocytes and adipocytes. These data indicated that the ob gene is expressed by preadipocytes and ob gene expression may be correlated with preadipocyte recruitment as well as fat cell size.     

Osteogenic protein-1 and interleukin-6 with its soluble receptor synergistically stimulate rat osteoblastic cell differentiation

Osteogenic Protein-1 (OP-1, BMP-7), a member of the bone morphogenetic protein family, stimulates synthesis of biochemical markers characteristic of the osteoblastic and chondrocytic phenotypes and induces new bone formation. Interleukin-6 (IL-6), a cytokine produced by a wide variety of cells, appears to interact with other factors producing different biological effects. In the present study, we showed that OP-1 action in fetal rat calvaria (FRC) cells was enhanced by the combination of IL-6 and the soluble receptor IL-6sR. OP-1 alone induced alkaline phosphatase (AP) activity by 4- to 5-fold above the control. Exogenous IL-6 soluble receptor (IL-6sR) synergistically stimulated the OP-1-induced AP activity and mineralized bone nodule formation by an additional 3-fold. The stimulation was IL-6sR concentration-dependent. The combination of IL-6 and IL-6sR synergistically stimulated OP-1 action by an additional 6- to 7-fold. BMPR-II receptor mRNA expression in FRC cells treated with OP-1 and IL-6 plus IL-6sR was stimulated further, while BMPR-IA, -IB, and ActR-I expressions were not affected. The intracellular signaling molecules Smad2 and Smad5 mRNA expressions were not changed under these conditions. The expression of selected BMP family members (BMP-3, -4, and -6) was altered in FRC cells treated with OP-1 in combination with IL-6 and IL-6sR. The combination of IL-6 and IL-6sR reduced the OP-1-stimulated BMP-3 mRNA levels and enhanced the suppressive effect of OP-1 on BMP-4 and -6 mRNA expressions. In conclusion, the present results demonstrate that exogenous IL-6 and IL-6sR synergistically stimulate OP-1 action in primary cultures of rat osteoblastic cells. One possible mechanism of synergy involves differential regulation of the effects of OP-1 on the expression of the type II BMP receptor and several other BMPs.     

Induction of perforin and serine esterases in a murine cytotoxic T lymphocyte clone

The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.