Human IL-1 Beta Research Articles

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.

Effects of carprofen and dexamethasone on canine chondrocytes in a three-dimensional culture model of osteoarthritis.

To determine effects of carprofen and dexamethasone on chondrocytes in a culture model of osteoarthritis (OA). Chondrocytes isolated from articular cartilage of the humeral head of 5 adult dogs. Chondrocytes were harvested, cultured and subcultured in monolayer, and then cultured in a 3-dimensional (3-D) medium. Cells from each dog were distributed into 6 groups with differing content of liquid medium for each 3-D construct (agarose [AG], AG plus interleukin [IL]-1beta, AG plus carprofen [4 microg/mL], AG plus dexamethasone [1 mg/mL], AG plus IL-1beta [20 ng/mL] plus carprofen [4 microg/mL], and AG plus IL-1beta (20 ng/mL) plus dexamethasone (1 mg/mL). On days 3, 6, 12, and 20 of culture, samples from all groups were collected. Liquid media were assayed for glycosaminoglycan, prostaglandin (PG)E2, matrix metalloprotease (MMP)-3, and MMP-13 concentrations. All 3-D constructs were evaluated for viability, cell morphology, proteoglycan staining, and collagen type-II concentration. Total glycosaminoglycan content in each 3-D construct was quantitated by spectrophotometric assay. Addition of IL-1beta caused a significant loss of cell viability and matrix production. Addition of carprofen or dexamethasone caused significant decreases in PGE2 in the liquid media, and each was minimally effective in protecting chondrocytes against negative effects of IL-1beta. Human recombinant IL-1beta resulted in loss of cell viability, alterations in extracellular matrix components, and production of PG and MMP Carprofen and dexamethasone had little effect on cell and matrix variables but did decrease PGE2 concentrations and primarily affected the inflammatory pathway of osteoarthritis.

Recombinant interleukin-1 beta activates the hypothalamic-pituitary-interrenal axis in rainbow trout, Oncorhynchus mykiss.

The present study provides the first direct evidence that implicates fish cytokines as the effector molecules by which the immune system signals the neuroendocrine system and activates the hypothalamic-pituitary-interrenal stress axis. I.p. injections of trout recombinant interleukin-1 beta (rIL-1 beta) or E. coli lipopolysaccharide (LPS), at concentrations known to induce immune/inflammatory responses in vivo (0.1-0.6 nmol/kg and 1.3 mg/kg respectively), significantly elevated plasma cortisol levels in a dose- and/or time-dependent manner. However, in contrast to general stress responses in fish, under the conditions employed in this study, no specific treatment effects on plasma glucose levels could be demonstrated. The trout IL-1 beta peptides (P1 and P3), which are homologous to receptor-binding sequences of human IL-1 beta, failed to influence the prevailing cortisol concentration even though an equivalent dose has been found to have immunostimulatory properties in vivo. Blockade of endogenous ACTH release by administration of the synthetic glucocorticoid dexamethasone prevented the rIL-1 beta/LPS-mediated elevation of plasma cortisol, suggesting that IL-1 beta and LPS modulate cortisol secretion via effects at the level of the hypothalamic-pituitary axis. These data indicate that, with respect to IL-1 beta, cytokine signalling between the immune and neuroendocrine systems in mammals appears to be conserved in lower vertebrates.

Proinflammatory cytokine IL-1 beta promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction.

Inflammatory conditions are associated with tumor development. IL-1beta is a multifunctional and proinflammatory cytokine that affects nearly all types of cells. To investigate the role of IL-1beta in tumor growth in vivo, we transduced the retroviral vector coding human IL-1beta gene into mouse Lewis lung carcinoma (LLC) cells and subsequently inoculated the transformant (LLC/IL-1beta) to syngeneic C57BL/6 mice. Tumors derived from LLC/IL-1beta grew faster (240%, day 18, vs null-vector control LLC/neo; p < 0.01) and showed more abundant vasculature (250%, vs LLC/neo; p < 0.05), whereas LLC/IL-1beta cells, LLC/neo cells, and wild-type LLC cells did not show any significant difference in the growth rate in vitro. As compared with LLC/neo cells, LLC/IL-1beta cells secreted 2-fold the amount of vascular endothelial growth factor and >10-fold the amount of macrophage-inflammatory protein-2 (CXCL2), one of whose main functions is angiogenesis. Although LLC/IL-1beta itself did not secrete hepatocyte growth factor (HGF), the tumor derived from LLC/IL-1beta cells also contained a >4-fold higher concentration of HGF, another angiogenic factor. In situ hybridization of HGF mRNA in LLC/IL-1beta tumor sections demonstrated that stromal fibroblasts and infiltrating cells overexpressed HGF mRNA. Moreover, when cultured in the presence of HGF in vitro, LLC/IL-1beta cells secreted even larger amounts of vascular endothelial growth factor and macrophage-inflammatory protein-2. The antiangiogenic agent TNP-470 and anti-CXCR2 Ab inhibited the tumor growth of LLC/IL-1beta cells in vivo. These results indicated that secreting IL-1beta into the tumor milieu induces several angiogenic factors from tumor and stromal cells and thus promotes tumor growth through hyperneovascularization.

Nuclear factor kappa B activation and regulation of cyclooxygenase type-2 expression in human amnion mesenchymal cells by interleukin-1beta.

Interleukin-1beta (IL-1beta) has been shown in numerous studies to increase prostaglandin (PG) output by up-regulating the expression of cyclooxygenase-2 (COX-2), a rate-limiting enzyme in PG synthesis. In this study, we investigated the possible role of the nuclear factor kappa B (NFkappaB) in IL-1beta signaling, leading to the expression of COX-2 in human amnion cell culture. Fetal amnion was obtained following vaginal delivery and digested with collagenase, and the subepithelial (mesenchymal) cells were isolated. Cultures were characterized with antisera to keratin (epithelial cells) and vimentin (mesenchymal cells). Confluent cells were stimulated with human recombinant IL-1beta, and activation of NFkappaB was assessed by measuring changes in the inhibitory protein IkappaB (total IkappaB and phosphorylated IkappaB) using Western blot analysis as well as by nuclear binding of NFkappaB using an electrophoretic mobility shift assay. COX-2 protein levels were determined by Western blot analysis. After 5 min of stimulation with IL-1beta, phosphorylated IkappaB began to appear, 90% of which was degraded within 15 min. This was temporally associated with decreased total IkappaB and increased nuclear NFkappaB DNA-binding activity. In the IL-1beta-treated group, COX-2 protein began to increase after 6 h; this response was time-dependent, with a significant increase until 24 h after IL-1beta stimulation. When NFkappaB translocation was blocked by using SN50 (a cell-permeable inhibitory peptide of NFkappaB translocation), the synthesis of COX-2 protein was inhibited. These results suggest that NFkappaB is involved in the IL-1beta-induced COX-2 expression in the mesenchymal cells of human amnion.

Differential modulation of synthesis of human il-1 beta and il-1 receptor antagonist by cyclosporin a

Differential modulation of synthesis of human il-1 beta and il-1 receptor antagonist by cyclosporin a

Fetal pig beta cells are resistant to the toxic effects of human cytokines.

The cytokine tumour necrosis factor-alpha (TNF-alpha) is thought to be responsible for primary nonfunction of islets when transplanted. This, and two other cytokines, interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) are also implicated in the autoimmune destruction of beta cells. It is unknown if the fetal pig beta cell, which is being transplanted as a treatment for type 1 diabetes, is affected by these cytokines. We compared the effects of the cytokines on the function and viability of adult and fetal pig beta cells. The cells were cultured for up to 3 days in the presence of 2000 pg/ml of human IL-1beta, 1000 U/ml of TNF-alpha, and 1000 U/ml of IFN-gamma, as well as 1000 U/ml of porcine IFN-gamma. Cumulative insulin levels, insulin content, metabolic activity, and viability of these cells were examined. Additionally, nitric oxide production and the activity of antioxidant enzymes in these cells were also determined. TNF-alpha and the combination of the three human cytokines caused a transient increase in cumulative insulin levels. TNF-alpha alone enhanced insulin content on day 3. There was no effect of these human cytokines on mitochondrial function and viability. In contrast, porcine IFN-gamma inhibited fetal pig beta cell function and also caused their death. Adult pig islets are sensitive to the toxic effects of human TNF-alpha, IL-1beta, the combination of the three cytokines, and porcine IFN-gamma. The activity of the antioxidant enzymes catalase, glutathione peroxidase, and superoxide dismutase were significantly higher in fetal pig beta cells than in adult islets, implying that this may be the reason for the lack of adverse effects of the cytokines on the fetal beta cell. Fetal pig beta cells are resistant to the toxic effect of the human cytokines, TNF-alpha and IL-1beta, in vitro. This resistance suggests that fetal, but not adult beta cells, when transplanted into humans with type 1 diabetes may be protected from primary nonfunction and will be partially protected from autoimmune destruction.

Effect of interleukin-18 on mouse core body temperature.

We have studied, using a telemetry system, the pyrogenic properties of recombinant murine interleukin-18 (rmIL-18) injected into the peritoneum of C57BL/6 mice. The effect of IL-18 was compared with the febrile response induced by human IL-1beta, lipopolysaccharide (LPS), and recombinant murine interferon-gamma (rmIFN-gamma). Both IL-1beta and LPS induced a febrile response within the first hour after the intraperitoneal injection, whereas rmIL-18 (10-200 microg/kg) and rmIFN-gamma (10-150 microg/kg) did not cause significant changes in the core body temperature of mice. Surprisingly, increasing doses of IL-18, injected intraperitoneally 30 min before IL-1beta, significantly reduced the IL-1beta-induced fever response. In contrast, the same pretreatment with IL-18 did not modify the febrile response induced by LPS. IFN-gamma does not seem to play a role in the IL-18-mediated attenuation of IL-1beta-induced fever. In fact, there was no elevation of IFN-gamma in the serum of mice treated with IL-18, and a pretreatment with IFN-gamma did not modify the fever response induced by IL-1beta. We conclude that IL-18 is not pyrogenic when injected intraperitoneally in C57BL/6 mice. Furthermore, a pretreatment with IL-18, 30 min before IL-1beta, attenuates the febrile response induced by IL-1beta.

Autocrine Up-Regulation of Glucocorticoid Receptors by Interleukin-6 in Human Osteoblast-Like Cells

Interleukin (IL)-6 is a bone-resorbing cytokine that acts primarily on osteoclast progenitors to stimulate both proliferation and differentiation. Glucocorticoids (GC) down-regulate IL-6 synthesis in different cell types, including osteoblasts. Given the fact that bone remodeling is a tightly controlled process, it is reasonable to think of auto-regulatory mechanisms in the bone microenvironment able to prevent excess IL-6 production. We have studied two human osteosarcoma cell lines (Saos-2 and MG-63) with different degrees of differentiation and different constitutive IL-6 production (3.4 +/- 0.2 (mean +/- SE) and 2,898 +/- 401 pg/10(6) cells, respectively). We measured the expression of glucocorticoid receptor (GR) in terms of specific binding sites after exposure of cells to different amounts of IL-6. Incubation for 20 hours with IL-6 at increasing concentrations up to 2,000 pg/ml yielded significant increase of GR binding sites in both cell lines. IL-6 was also able to revert the inhibitory effect of dexamethasone (1 microM) on GR in both cell lines. In MG-63 cells, that express higher concentrations of GR, IL-6 deprivation via a specific anti-IL-6 antibody (100 ng/ml) significantly decreased GR, as it was noticed, although to a lesser degree, using a specific anti-IL-6 receptor antibody. In Saos-2, cells that express lower concentrations of GR, a 40-hour treatment with human IL-1beta (10 ng/ml) significantly increased both IL-6 production and GR. This latter effect was completely abolished by co-treating the cells with the anti-IL-6 antibody. Our data are consistent with an autocrine up-regulation of GR expression by IL-6 in human osteoblast-like cells. This phenomenon, which is also relevant to paracrine cell-to-cell communication, subserves a feedback loop in the bone microenvironment that restrains excess inducible IL-6 production. In patients having high levels of IL-6 and given GCs, it could offer an additional explanation for the biphasic pattern of bone loss in the course of therapy.

HIV-1 coat protein gp120 stimulates interleukin-1beta secretion from human neuroblastoma cells: evidence for a role in the mechanism of cell death.

1. The role of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) in the mechanism of cell death induced by the human immunodeficiency virus type 1 (HIV-1) recombinant coat glycoprotein, gp120 IIIB, has been studied in the human CHP100 neuroblastoma cell line maintained in culture. 2. Death of neuroblastoma cells typically elicited by 10 pM gp120 or by human recombinant IL-1beta (10 ng x ml(-1)) has been minimized by the antagonist of IL-1 receptor, i.e. IL-1ra (0.5 and 50 ng x ml(-1), respectively), an endogenous molecule that antagonizes most of the biological actions of IL-1beta, or by an antibody (5 and 50 ng x ml(-1)) which blocks the human IL-1 receptor type I (IL-1RI). 3. ELISA experiments have established that gp120 enhances immunoreactive IL-1beta levels in the culture medium and this is prevented by exposure to the IL-1 converting enzyme (ICE) inhibitor t-butoxycarbonyl-L-aspartic acid benzyl ester-chloromethylketone [Boc-Asp(OBzl)-CMK] used at a concentration (2.5 microM) which significantly (P<0.001) reduces cell death. 4. Death of CHP100 cells induced by gp120 is also prevented by acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK; 10-100 microM), a second inhibitor of ICE, supporting the concept that the viral protein stimulates the conversion of the 31 kDa pro-IL-1beta in to the 17 kDa mature cytokine which is then secreted to cause death. 5. In conclusion, our present data demonstrate that gp120 stimulates the secretion of IL-1beta which then triggers CHP100 neuroblastoma cell death via stimulation of IL-1 receptor type I.

Differential effects of estradiol on the adrenocorticotropin responses to interleukin-6 and interleukin-1 in the monkey.

Endotoxin and the inflammatory cytokines interleukin (IL)-1 and IL-6 are potent activators of the hypothalamic-pituitary-adrenal (HPA) axis. Although estradiol (E(2)) has been shown to enhance the HPA response to certain types of stress, previous studies in the rodent have shown that HPA responses to endotoxin and to IL-1 were enhanced by ovariectomy and attenuated by E(2). The mechanisms underlying these observations are unclear, but there is evidence that E(2) may have direct inhibitory effects on IL-6 synthesis and release. Because endotoxin and IL-1 both stimulate IL-6, it is possible that the E(2)-induced suppression of the HPA response to endotoxin and IL-1 results from decreased IL-6 release. We have therefore examined the ACTH response to IL-6 and IL-1beta in six ovariectomized rhesus monkeys with and without 3 weeks of E(2) replacement. In the first study, plasma ACTH levels peaked at 60 min after iv injection of 6 microg recombinant human IL-6. Both the ACTH response, over time, and the area under the ACTH response curve were significantly higher in the E(2)-treated animals (P < 0.05). The peak ACTH level was 66 +/- 16 pg/ml without E(2) vs. 161 +/- 69 pg/ml with E(2). In the second study, iv infusion of recombinant human IL-1beta (400 ng) produced plasma IL-6 levels comparable with those seen after IL-6 injection in the first study. In the IL-1 study, however, there was a significant attenuation of the ACTH response, over time, in the E(2)-treated animals (P < 0.001); the peak ACTH level was 83 +/- 34 pg/ml vs. 13 +/- 4.4 pg/ml after E(2). The IL-6 response was similarly attenuated (P < 0.001); the peak IL-6 level was 614 +/- 168 pg/ml vs. 277 +/- 53 pg/ml after E(2) treatment. Our results demonstrate that physiological levels of E(2) enhance the ACTH response to IL-6 but attenuate the ACTH response to IL-1. The attenuated ACTH response to IL-1 was accompanied by a blunted IL-6 response. Our results suggest that the blunted HPA response to IL-1 can be explained, at least in part, by E(2)-induced alterations in IL-6 release. It remains to be determined whether E(2) affects other inflammatory mediators that also participate in this process.

Structural and Functional Similarity between Yersinia pestis Capsular Protein Caf1 and Human Interleukin-1β

A comparative study of the structural and functional properties of recombinant Yersinia pestis Caf1 and human IL-1beta was performed. According to Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) data, IL-1beta and Caf1 are typical beta-structural proteins. Neither protein interacts with the hydrophobic probe ANS (8-anilino-1-naphthalenesulfonate) under physiological conditions. Specific binding of Caf1 [K(d) = (5.4 +/- 0.1) x 10(-10) M] to interleukin-1 receptors (IL-1Rs) on the surface of finite mouse fibroblasts (line NIH 3T3) was observed. Caf1 is able to inhibit high-affinity binding of (125)I-labeled IL-1beta to NIH 3T3 cells, and in the presence of Caf1, the binding of [(125)I]IL-1beta is characterized by a K(d) of (2.0 +/- 0.3) x 10(-9) M. Caf1 binding to IL-1R could reflect adhesive properties of the capsular subunits responsible for the contact of bacteria with the host immunocompetent cells. In its turn, this may represent a signal for the initiation of the expression and secretion of the proteins of Y. pestis Yop virulon. Thus, these results help to explain the importance of Caf1 in the interaction of Y. pestis with the host immune system.

Interleukin (IL)-1beta regulation of IL-1beta and IL-1 receptor antagonist expression in cultured human endometrial stromal cells.

The interleukin (IL)-1 system is a major regulator of local cellular interactions during embryonic implantation. Because IL-1beta and IL receptor antagonist (IL-1ra) are both expressed in human endometrium, we hypothesized that an appropriate ratio of IL-1beta to IL-1ra might favor the process of embryo implantation. Therefore, we investigated IL-1 regulation of the quantitative ratio of IL-1beta/IL-1ra messenger RNA (mRNA) expression in human endometrial stromal cells using quantitative competitive PCR, as well as intracellular protein expression after stromal cell solubilization. Confluent stromal cell cultures were stimulated with human IL-1beta (0-1000 IU/mL) for 24 h. After 24 h, total RNA was extracted, reverse transcribed, and coamplified by PCR with a defined amount of internal standard. The quantitative ratio was determined by the density of target to the internal standard. After culture with IL-1beta for 24 and 48 h, stromal cells were solubilized, and the intracellular protein levels of IL-1beta and IL-1ra were measured by enzyme-linked immunosorbent assay. The IL-1beta and IL-1ra mRNA were both up-regulated, and IL-1R tI mRNA was down-regulated, by IL-1beta in a dose-dependent manner. The quantitative ratio of IL-1beta to IL-1ra mRNA was constant with the presence of increasing concentrations of IL-1beta (1-1000 IU/mL). IL-1beta and IL-1ra protein was not detected in conditioned media of cultures before addition of IL-1beta. IL-1beta and IL-1ra protein levels increased with increasing amounts of IL-1beta after solubilization of stromal cells. The IL-1beta was detectable after 12 h of culture, in comparison with IL-1ra, which was detectable after 24 h of IL-1beta stimulation. These results suggest that IL-1 may play a crucial role in embryo-maternal interaction by regulating stromal cell expression of IL-1beta and IL-1ra, resulting in an appropriate ratio during the process of embryonic implantation.

Expression of interleukin-18 and its monokine-directed function in rheumatoid arthritis.

To investigate the expression of and monokine induction by interleukin 18 (IL-18; also called interferon-gamma inducing factor, IGIF), in peripheral blood mononuclear cells (PBMC) and cultured synoviocytes from rheumatoid arthritis (RA) patients. We carried out IL-18 Western blotting and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) of cytokines in PBMC [IL-18, IL-1beta and tumour necrosis factor alpha (TNF-alpha)] and long-term cultured fibroblast-like synoviocytes (FLS) [IL-18, IL-1beta, TNF-alpha, IL-6, interferon gamma (INF-gamma) and [granulocyte-macrophage colony stimulating factor (GM-CSF)] from RA patients and controls. FLS were isolated from RA synovial membranes (FLS(SM)) and RA synovial fluids (FLS(SF)), osteoarthritis (OA) FLS(SM) and FLS(SF) from spondyloarthropathy patients. FLS were characterized by fluorescence-activated cell sorting of the FLS. PBMC and FLS from RA patients and control subjects were stimulated with recombinant human IL-18 and IL-1beta (rHuIL-18/rHuIL-1beta), and TNF-alpha, IL-1beta and MMP-1 were measured by ELISA in supernatants. Constitutive expression of IL-18 mRNA was significantly reduced whereas that of TNF-alpha was enhanced in RA PBMC. Persistent low expression of IL-18, TNF-alpha, GM-CSF and IL-1beta was observed in RA and OA FLS(SM) as well as spondyloarthropathy FLS(SF). In contrast, high constitutive expression of IL-18 in FLS (CD90/Thy-1- and CD54-positive, CD14- and CD86-negative), accompanied by persistent high levels of TNF-alpha, GM-CSF and IL-1beta expression, was restricted to synovial fluid-derived FLS obtained from RA patients. IFN-gamma was not detectable in any culture, but IL-6 mRNA was equally expressed in all FLS cultures. rHuIL-18 was effective in stimulating TNF-alpha and IL-1beta secretion in PBMC from healthy controls, but failed to stimulate TNF-alpha and IL-1beta secretion from PBMC in 11 of 12 RA patients, and all FLS cultures. rHu-IL-1beta, but not rHu-IL-18, induced interstitial collagenase (MMP-1) in FLS. Persistent high production of proinflammatory cytokines in RA-FLS(SF) may be relevant for chronic progression in RA synovitis. Levels of TNF-alpha and IL-1beta expression are increased in RA-FLS(SF), but are independent of IL-18. The pathological function of enhanced IL-18 expression in RA-FLS(SF) remains to be further elucidated.

Insertion of the DNA for the 163-171 peptide of IL1beta enables a DNA vaccine encoding p185(neu) to inhibit mammary carcinogenesis in Her-2/neu transgenic BALB/c mice.

An assessment was made of the effectiveness of DNA vaccination in prevention of the mammary adenocarcinomas of BALB/c female mice transgenic for the activated rat Her-2/neu oncogene. Atypical hyperplasia is evident in their mammary glands when they are 6 weeks old and in situ carcinoma by the 13th week. Palpable invasive carcinomas appear around the 17th week and are always evident in all 10 glands by the 33rd week. Intramuscular vaccinations with 100 microg plasmid DNA encoding the extracellular domain of the Her-2/neu p185 (ECD) performed at the 6th, 12th, 18th and 24th week provided no significant protection, whereas those ECD plasmids in which the DNA coding for the immunomodulatory 163-171 (VQGEESNDK) nonapeptide of human IL1beta (ECD-IL1betap) had been inserted both delayed carcinogenesis and reduced tumor multiplicity. This reduction was associated with a marked immune-inflammatory reaction and a conspicuous leukocyte infiltrate located in the stroma surrounding the hyperplastic mammary ductul-alveolar structures. It was also directly correlated with a high anti-p185(neu) antibody production and an immunoglobulin switch to IgG2a and IgA. No anti-p185(neu) cytotoxic response was found. No significant protection was obtained when the DNA coding for the non-active peptide 189-197 of IL1beta was inserted.

Interleukin-1beta gene transcription in U937 cells is modulated by type I collagen and cytoskeletal integrity via distinct signaling pathways.

Type I collagen (Col), an extracellular matrix molecule highly expressed in injured tissues, stimulates interleukin-1beta (IL-1beta) expression in monocytic cells. Using U937 cells transfected with the human IL-1beta gene promoter connected to a reporter gene, we examined how the organizational state of the cytoskeleton modulates the expression of IL-1beta after Col stimulation. We found the same degree of stimulation of IL-1beta gene transcription in cells exposed to Col presented in different fashions (i.e., soluble Col, Col-coated plate, three-dimensional Col lattice), suggesting that stimulation of IL-1beta is independent of the mode of presentation of Col. The Col-stimulated response was associated with induction of the transcription factor activator protein-1 (AP-1) and was abolished by a protein kinase C (PKC) inhibitor, by a mitogen-activated protein kinase (MAPK) inhibitor, and by cotransfection of cells with a competing AP-1 oligo. Disruption of cytoskeletal organization with colchicine or cytochalasin B stimulated IL-1beta gene transcription and enhanced the cells' response to Col. The effects of cytochalasin and colchicine were inhibited by the PKC inhibitor but were not affected by the MAPK inhibitor or the AP-1 oligo. These findings suggest that the cytoskeletal integrity modulates the constitutive and Col-stimulated transcription of the IL-1beta gene via distinct signaling mechanisms.

Statins prevent endothelial cell activation induced by antiphospholipid (anti-beta2-glycoprotein I) antibodies: effect on the proadhesive and proinflammatory phenotype.

To investigate the ability of statins, the inhibitors of the hydroxymethylglutaryl-coenzyme A reductase enzyme, to affect endothelial cell activation induced by anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in vitro. Human umbilical vein endothelial cell (HUVEC) activation was evaluated as U937 monocyte adhesion, E-selectin, and intercellular adhesion molecule I (ICAM-1) expression by cell enzyme-linked immunosorbent assay and as interleukin-6 (IL-6) messenger RNA (mRNA) expression by RNA protection assay. E-selectin-specific nuclear factor kappaB (NF-kappaB) DNA-binding activity was evaluated by the gel-shift assay. HUVECs were activated by polyclonal affinity-purified IgG, human monoclonal IgM anti-beta2GPI antibodies, human recombinant IL-1beta, tumor necrosis factor alpha, or lipopolysaccharide (LPS). Fluvastatin reduced, in a concentration-dependent manner (1-10 microM), the adhesion of U937 to HUVECs and the expression of E-selectin and ICAM-1 induced by anti-beta2GPI antibodies as well as by cytokines or LPS. Another lipophilic statin, simvastatin, displayed similar effects but to a lesser extent than fluvastatin. The inhibition of E-selectin expression exerted by fluvastatin was related to the impairment of NF-kappaB binding to DNA. Moreover, the drug attenuated the expression of IL-6 mRNA in HUVEC exposed to anti-beta2GPI antibodies or cytokines. Incubation of HUVECs with mevalonate (100 microM), concomitantly with fluvastatin, greatly prevented the inhibitory effect of statin. Endothelial activation mediated by anti-beta2GPI antibody can be inhibited by statins. Because of the suggested role of endothelial cell activation in the pathogenesis of antiphospholipid syndrome (APS), our data provide, for the first time, a rationale for using statins as an additional therapeutic tool in APS.

Molecular and cellular mediators of interleukin-1-dependent acute inflammatory arthritis.

To examine the molecular and cellular mechanisms in a model of acute inflammatory monarticular arthritis induced by methylated bovine serum albumin (mBSA) and interleukin-1 (IL-1). Mice were injected intraarticularly with mBSA on day 0 and subcutaneously with recombinant human IL-1beta on days 0-2. At day 7, knee joints were removed and assessed histologically. Flow cytometry and RNase protection were used to analyze IL-1-dependent events. C57BL/6 (B6), 129/Sv, and (B6 x 129/ Sv)F1 hybrid mice, all H-2b strains, were susceptible to mBSA/IL-1-induced arthritis, whereas C3H/HeJ (H-2k) mice were not. B6 mice lacking T and B cells (RAG1-/-) or major histocompatibility complex (MHC) class II antigens (MHCII-/-), and B6 mice treated with a CD4+ T cell-depleting monoclonal antibody, were resistant to disease. In contrast, B cell-deficient (muMT/ muMT) mice developed arthritis at an incidence and severity similar to that of controls. RelB-deficient (RelB-/-) bone marrow chimeric mice had arthritis that was significantly reduced in incidence and severity. In B6 mice, flow cytometry demonstrated an IL-1-dependent leukocyte infiltration into the synovial compartment and RNase protection assays revealed induction of messenger RNA (mRNA) for the chemokines monocyte chemoattractant protein 1, macrophage inhibitory protein 2 (MIP-2), RANTES, MIP-1alpha, and MIP-1beta, in vivo and in vitro. Arthritis induced by mBSA/IL-1 is strain specific and dependent on CD4+ T lymphocytes and at least partially on RelB, but not on B lymphocytes or antibody. IL-1 contributes to leukocyte recruitment to the synovium and directly induces chemokine mRNA production by synovial cells. This model of acute monarticular arthritis is particularly suitable for further investigations into cell-mediated immunity in arthritis and the role of IL-1.

Macrophages exposed continuously to lipopolysaccharide and other agonists that act via toll-like receptors exhibit a sustained and additive activation state

BackgroundMacrophages sense microorganisms through activation of members of the Toll-like receptor family, which initiate signals linked to transcription of many inflammation associated genes. In this paper we examine whether the signal from Toll-like receptors [TLRs] is sustained for as long as the ligand is present, and whether responses to different TLR agonists are additive.ResultsRAW264 macrophage cells were doubly-transfected with reporter genes in which the IL-12p40, ELAM or IL-6 promoter controls firefly luciferase, and the human IL-1β promoter drives renilla luciferase. The resultant stable lines provide robust assays of macrophage activation by TLR stimuli including LPS [TLR4], lipopeptide [TLR2], and bacterial DNA [TLR9], with each promoter demonstrating its own intrinsic characteristics. With each of the promoters, luciferase activity was induced over an 8 hr period, and thereafter reached a new steady state. Elevated expression required the continued presence of agonist. Sustained responses to different classes of agonist were perfectly additive. This pattern was confirmed by measuring inducible cytokine production in the same cells. While homodimerization of TLR4 mediates responses to LPS, TLR2 appears to require heterodimerization with another receptor such as TLR6. Transient expression of constitutively active forms of TLR4 or TLR2 plus TLR6 stimulated IL-12 promoter activity. The effect of LPS, a TLR4 agonist, was additive with that of TLR2/6 but not TLR4, whilst that of lipopeptide, a TLR2 agonist, was additive with TLR4 but not TLR2/6. Actions of bacterial DNA were additive with either TLR4 or TLR2/6.ConclusionsThese findings indicate that maximal activation by any one TLR pathway does not preclude further activation by another, suggesting that common downstream regulatory components are not limiting. Upon exposure to a TLR agonist, macrophages enter a state of sustained activation in which they continuously sense the presence of a microbial challenge.

Interleukin-6 upregulates glucocorticoid receptor numbers in human osteoblast-like cells.

Interactions between the endocrine and immune systems are well known with special regard to the hypothalamic-pituitary-adrenal axis. Little of the literature focuses on the complex effects of cytokines on tissue responsiveness to glucocorticoids (GC). In bone tissue, osteoblasts represent a valuable model for studying GC-cytokine interactions. Hence, we have studied two human osteosarcoma cell lines (Saos-2 and MG-63) with different degrees of differentiation and different constitutive IL-6 production (3.4 +/- 0.3 (mean +/- SE) and 3309 +/- 578 pg/10(6) cells). We measured glucocorticoid receptor (GR) number and affinity as a function of the exposure of cells to different amounts of IL-6. Incubation for 20 h with IL-6 at increasing concentrations up to 2000 pg/ml yielded a significant increase of GR binding sites in both cell lines. IL-6 was also able to reverse the inhibitory effect of dexamethasone (1 mumol/l) on GR in both cell lines. In MG-63 cells, expressing higher concentrations of GR, IL-6 deprivation via a specific anti-IL-6 antibody (100 ng/ml) significantly decreased GR. In Saos-2 cells, expressing lower concentrations of GR, a 40 h treatment with human IL-1 beta (10 ng/ml) significantly increased both IL-6 production and GR. This latter effect was completely abolished by co-treating the cells with the anti-IL-6 antibody. Our results provide evidence that IL-6 is an autocrine positive modulator of GR number in human osteoblasts.