Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P05231 (interleukin-6)
 23,907 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Titanium-aluminum-vanadium wear particles isolated from the soft-issue membrane of a failed total hip arthroplasty were added to human fibroblasts in cell culture. The cellular response to particle challenge was determined by assaying for levels of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, prostaglandin E2, basic fibroblast growth factor, platelet-derived growth factor-AB, and transforming growth factor-beta. Collagenase and gelatinase activities were analyzed by zymography and [3H]collagen degradation. Cell viability was assessed by measuring the uptake of [3H]thymidine. Over the range of particle concentrations tested, cell viability, as demonstrated by [3H]thymidine uptake, remained unaffected. Fibroblasts exhibited a dose-dependent release of interleukin-6 in response to exposure to titanium-aluminum-vanadium particles. At 6 and 48 hours, the highest concentration of titanium alloy particles (0.189% [vol/vol]) resulted in 7-fold and 16-fold increases in interleukin-6 release, respectively, when compared with negative controls. Neither interleukin-1 beta nor tumor necrosis factor-alpha was detected in the culture medium at any particle concentration tested for both dermal and foreskin fibroblasts. The pattern of prostaglandin E2 release by fibroblasts mirrored the pattern of interleukin-6 release. Fibroblasts exposed to the highest concentration of titanium alloy particles showed an increase in collagenase activity, starting at 12 hours. When medium samples were treated with amino phenylmercuric acetate to activate latent enzymes, a statistically significant increase in collagenase activity was observed as early as 6 hours (p < 0.001). Substrate gel analysis of medium from fibroblasts stimulated by high particle concentrations also showed an increase in gelatinolytic activity when compared with unstimulated controls. Analysis of medium samples for growth factors showed an increase in basic fibroblast growth factor at low particle concentrations, beginning at 12 hours. Levels of platelet-derived growth factor-AB and transforming growth factor-beta were not detectable in the controls or at any particle concentration tested. The results of this study showed that fibroblasts exposed to titanium alloy wear particles become activated and release proinflammatory mediators that influence bone metabolism. These data support the hypothesis that direct activation of fibroblasts by particulate wear may play a role in particle-mediated osteolysis. Fibroblast activation coupled with the biologic response of macrophages to wear debris in the loosening membrane may have a synergistic effect on pathologic bone resorption.
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PMID:In vitro activation of human fibroblasts by retrieved titanium alloy wear debris. 867 60

Contact of various cells with extracellular matrix molecules modulates their cellular functions and phenotype. Most investigations have employed dishes coated with purified matrix constituents or plain collagen I lattices omitting the effects of other important matrix components such as proteoglycans. In this study we analyze the effect of purified glycosaminoglycans (GAGs) on human fibroblasts and human umbilical vein endothelial cells (HUVEC) embedded within collagen I/III lattices. HUVEC contracted collagen I/III gels far less efficiently than fibroblasts and addition of heparan sulfate and heparin almost completely inhibited contraction. In collagen gels HUVEC down-regulated collagenase mRNA while increasing collagen I, IV mRNA expression. Addition of heparin and heparan sulfate reversed the collagen IV mRNA induction whereas hyaluronic acid and chondroitin sulfate enhanced fibronectin and collagenase transcripts. Fibroblasts readily contracted collagen gels, and mRNA levels for fibronectin, collagenase and interleukin-6 were stimulated. Gel contraction was mostly unaffected by the different glycosaminoglycans. Fibroblasts responded to the addition of dermatan sulfate, heparan sulfate and heparin with a decrease in fibronectin, collagenase and interleukin-6 mRNA. Binding studies revealed saturable binding sites on fibroblasts and HUVEC for 35S-labelled heparin, demonstrating specificity for heparin and heparan sulfate over other GAGs in competition experiments. This study implies that glycosaminoglycans participate in cell-matrix interactions by effectively modulating the cellular phenotype via high affinity binding sites.
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PMID:Glycosaminoglycans modulate cell-matrix interactions of human fibroblasts and endothelial cells in vitro. 883 71

Interleukin-6 (IL-6), a cytokine produced by skeletal cells, increases bone resorption, but its effects on collagenase expression are unknown. We tested the effects of IL-6 and its soluble receptor on collagenase 3 expression in osteoblast-enriched cells from fetal rat calvariae (Ob cells). IL-6 caused a small increase in collagenase mRNA levels, but in the presence of IL-6-soluble receptor (IL-6sR), IL-6 caused a marked increase in collagenase transcripts after 2-24 h. In addition, IL-6sR increased collagenase mRNA when tested alone. IL-6 and IL-6sR increased immunoreactive collagenase levels. Cycloheximide and indomethacin did not prevent the effect of IL-6 and IL-6sR on collagenase mRNA levels. IL-6 and IL-6sR did not alter the decay of collagenase mRNA in transcriptionally arrested Ob cells and increased the levels of collagenase heterogeneous nuclear RNA and the rate of collagenase gene transcription in Ob cells. IL-6 and IL-6sR increased collagenase 3 mRNA in MC3T3 cells but only modestly in skin fibroblasts. IL-6 and IL-6sR enhanced the expression of tissue inhibitor of metalloproteinases 1. In conclusion, IL-6, in the presence of IL-6sR, increases collagenase 3 synthesis in osteoblasts by transcriptional mechanisms. This effect may contribute to the action of IL-6 on bone matrix degradation and bone resorption.
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PMID:Interleukin-6 and its soluble receptor cause a marked induction of collagenase 3 expression in rat osteoblast cultures. 911 85

The response of human T lymphocytes to various stimuli includes the expression of the matrix metalloproteinase (MMP) genes stromelysin 2, gelatinase A and gelatinase B. The proteins encoded by these genes could confer the capacity to degrade macromolecular components of the extracellular matrix (ECM), and to shed transmembrane proteins such as tumor necrosis factor (TNF), TNF receptor, Interleukin-6 receptor and Fas ligand. To identify further MMP genes transcribed in T lymphocytes exposed to phorbol 12-myristate 13-acetate and a calcium ionophore, we combined reverse transcription and polymerase chain reaction using primers specific for conserved domains and detected collagenase 3 transcripts, first described in a human breast cancer. However, when the sequence of the complementary DNA was compared, additional 23 nucleotides were found in the 5' nontranslated region of the lymphocyte messenger RNA (mRNA). Northern blot analysis revealed 2 major inducible mRNA species of 1.9 and 2.8 kilobases, whose levels were lower than those of stromelysin 2. The observation that activated T lymphocytes transcribe several MMP genes, including a collagenase, indicates that the effector functions of these cells include enzymatic activities towards most constituents of the ECM, as well as some transmembrane proteins relevant to inflammation and apoptosis.
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PMID:A matrix metalloproteinase gene expressed in human T lymphocytes is identical with collagenase 3 from breast carcinomas. 956 63

The in-vitro effects of avocado and soybean unsaponifiable residues on neutral metalloproteinase activity, cytokines and prostaglandin E2 (PGE2) production by human articular chondrocytes were investigated. Avocado and soybean unsaponifiable residues were mixed in three ratios: 1:2 (A1S2), 2:1 (A2S1) or 1:1 (A2S2). Freshly isolated human chondrocytes were cultured for 72 h in the absence or presence of interleukin-1beta, (IL-1beta) (17 ng/ml), with or without unsaponifiable residue mixtures at a concentration of 10 microg/ml. A/S unsaponifiable residues were also tested separately at concentrations of 3.3, 6.6 and 10 microg/ml. All A/S unsaponifiable mixtures reduced the spontaneous production of stromelysin, interleukin-6 (IL-6), interleukin-8 (IL-8) and prostaglandin E2 (PGE2) by chrondrocytes. At concentrations of 3.3 and 6.6 microg/ml, A/S residues, tested separately, were potent inhibitors of the production of IL-8 and PGE2. Nevertheless, only avocado residue inhibited IL-6 production at these concentrations. A/S unsaponifiable mixtures had a more pronounced inhibitory effect on cytokine production than avocado or soybean residues added alone. As anticipated, IL-1beta induced a marked release of collagenase, stromelysin, IL-6, IL-8 and PGE2. A/S unsaponifiable mixtures partially reversed the IL-1 effects on chrondrocytes. These findings suggest a potential role for A/S unsaponifiable extracts in mitigating the deleterious effects of IL-1beta: on cartilage.
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PMID:Effects of three avocado/soybean unsaponifiable mixtures on metalloproteinases, cytokines and prostaglandin E2 production by human articular chondrocytes. 958 76

Cyclosporine A is a powerful immunosuppressive agent which is widely used for the prevention of allograft rejection and for the treatment of autoimmune diseases. Clinical and experimental data show that it may also act on connective tissue. We investigated the influence of cyclosporine A on granulation tissue formation and wound healing. Using an in vitro approach, we followed the time course of rat dermal fibroblasts during wound repair. Granulation fibroblasts were compared to dermal fibroblasts flow cytometrically and by mRNA analysis with respect to the expression of procollagen alpha1(I), integrin beta1, interleukin-6, transforming growth factor beta1, keratinocyte growth factor and activin betaA. The most pronounced effect in cyclosporine-treated rats was the strong down-regulation of activin beta expression. In cryo-sections of granulation tissue from the same rats, the distribution and expression intensity of intercellular adhesion molecule and its receptors were investigated by immunohistology. Clearly, a time course was detectable. Tissue from CsA-treated animals showed a delay of three days compared to untreated animals. Apoptosis was also delayed in CsA-treated rats by around three days. Furthermore, we investigated the effect of CsA on the expression of collagen alpha1(I), fibronectin and matrix metalloprotease 1 genes in dermal fibroblasts from untreated donors. No changes in the mRNA steady state levels of these genes were revealed after direct addition of different doses of CsA to fibroblast cultures. Our data suggest that CsA may interfere with the complicated net of interactions between connective tissue and the immune system by down-regulation of the inflammatory phase by modulation of cytokines and a subsequent delay of tissue repair.
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PMID:Cyclosporine A delays wound healing and apoptosis and suppresses activin beta-A expression in rats. 964 3

1. Hepatic stellate cells are key mediators of hepatic fibrosis. We have studied hepatic stellate cell expression of the collagenase and general protease inhibitor alpha2-macroglobulin after activation in tissue culture and in response to certain cytokines. 2. Hepatic stellate cells isolated by Pronase-collagenase digestion were activated by culture on uncoated plastic. By Northern analysis hepatic stellate cells undergoing activation (5 days) expressed alpha2-macroglobulin mRNA and alpha2-macroglobulin could be immunolocalized to hepatic stellate cells from 5 to 15 days of culture. 3. By ELISA of cell culture supernatants hepatic stellate cell secretion of alpha2-macroglobulin was found to increase from 2. 78+/-1.13 ng x ml-1 x microgram-1 DNA per 24 h at 5 days of culture (n=8) to 13.55+/-4.64 ng x ml-1 x microgram-1 DNA per 24 h at 15 days of culture (n=7). Stimulation of hepatic stellate cells with interleukin-6 at 5 days caused a significant increase in alpha2-macroglobulin expression as did exposure to Kupffer-cell conditioned medium. However, exposure of hepatic stellate cells to interleukin-1, transforming growth factor-beta1 and tumour necrosis factor-alpha had no significant effect. 4. During profibrotic liver injury plasma alpha2-macroglobulin levels were found to increase to between 850% and 250% of the control value (100%) after bile duct ligation (72 h to 13 days respectively), and to 1166% and 1106% of the control value during progressive CCl4-induced fibrosis (24 h to 4 weeks respectively). 5. These data suggest that hepatic stellate cells are a potential source of the potent protease inhibitor alpha2-macroglobulin, expression of which may inhibit matrix remodelling during progressive fibrosis.
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PMID:Rat hepatic stellate cell expression of alpha2-macroglobulin is a feature of cellular activation: implications for matrix remodelling in hepatic fibrosis. 968 May

We recently reported on the successful generation of immortalized (CEPI-17-CL4) cells from primary human corneal epithelial (P-CEPI) cells which exhibited phenotypic, immunohistochemical and metabolic characteristics akin to the P-CEPI cells. The aims of the present studies were to investigate the ligand binding and functional coupling of the histamine receptors to various biochemical and physiological systems in the P-CEPI and CEPI-17-CL4 cells and to relate these findings to the normal and/or pathophysiological role of histamine on the human ocular surface. Specific [3H]-pyrilamine binding to CEPI-17-CL4 cell homogenates comprised >93% of the total binding and represented interaction with an apparent single population of high affinity (Kd=3.76+/-0.78 nM; n=4) and saturable (Bmax = 1582+/-161 fmol g(-1) tissue) number of histamine-1 (H1) receptor binding sites on CEPI-17-CL4 cell homogenates. The H1-receptor selective antagonists, pyrilamine (Ki=3.6+/-0.84 nM, n=4) and triprolidine (Ki = 7.7+/-2.6 nM, n=3), potently displaced [3H]-pyrilamine binding, while the H2- and H3-receptor selective antagonists, ranitidine and clobenpropit, were weak inhibitors (K(i)s>13 microM). Histamine induced phosphoinositide (PI) hydrolysis 2.7-4.4 fold above basal levels and with a potency of 14.9+/-4.9 microM (n=9) and 4.7+/-0.2 microM (n=9) in P-CEPI and CEPI-17-CL4 cells, respectively. Histamine-induced PI turnover was antagonized by H1-receptor selective antagonist, triprolidine, with a potency (Ki) of 3.2+/-0.66 nM (n=10) and 3.03+/-0.8 nM (n=4) in P-CEPI and CEPI-17-CL4 cells, respectively, but weakly effected by 10 microM cimetidine and clobenpropit, H2- and H3-receptor antagonists. The PI turnover response was attenuated by pre-treatment of the cells with the selective phospholipase C inhibitor, U73122 (1-(6-((17beta-3-methoxyestra- 1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (IC50=4.8+/-2.4 microM, n = 3). Histamine stimulated intracellular Ca2+ ([Ca2+]i) mobilization in CEPI-17-CL4 cells with a potency of 6.3+/-1.5 microM (n=4). The histamine-induced [Ca2+]i mobilization was reduced by about 28% following pre-incubation of the cells with 4 mM EGTA. While triprolidine completely inhibited histamine-induced [Ca2+]i mobilization, it did not influence the bradykinin-induced [Ca2+]i mobilization response. Histamine (EC50s = 1.28-2.77 microM, n=3-4) concentration-dependently stimulated the release of interleukin-6 (IL-6), IL-8 and granulocyte macrophage colony-stimulating factor, but it did not significantly alter release of tumour necrosis factor-alpha, PGE2 or collagenase-1 (matrix metalloproteinase-1; MMP-1) from CEPI cells. However, IL-1 (10 ng ml(-1)), foetal bovine serum (10%) and phorbol-12-myristate-13-acetate (3 microg ml(-1)) were effective positive control secretagogues of all the cytokines, PGE2 and MMP-1, respectively, from these cells. It is concluded that the CEPI cells express H1-histamine receptors which are positively coupled to PI turnover and [Ca2+]i mobilization which may be directly or indirectly responsible for the release of various cytokines from these cells at physiologically and/or pathologically relevant concentrations.
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PMID:Pharmacology of [3H]-pyrilamine binding and of the histamine-induced inositol phosphates generation, intracellular Ca2+ -mobilization and cytokine release from human corneal epithelial cells. 986 65

Although parathyroid hormone (PTH) has the ability to stimulate bone growth in both rats and humans, its mechanism of action is not fully understood at the molecular level. An in vitro marker that reflects the in vivo anabolic actions of PTH would facilitate the discovery of small-molecule compounds that stimulate bone growth. We therefore compared the patterns of gene expression in three cell lines treated with PTH. The levels of c-fos, collagenase, interleukin-6 (IL-6), and collagen mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) in three osteoblast-like cell lines. The most responsive marker was c-fos, which was induced 5-10-fold after 1 h of PTH treatment in the UMR106-01 cell line. Because it is a critical early response gene in bone growth, we investigated the possibility of using c-fos stimulation as a method to screen for compounds that can stimulate bone formation. A highly sensitive, medium-throughput RT-PCR assay for c-fos mRNA expression was established using the Taqmantrade mark Detection System (Perkin Elmer, Mississauga, Ontario). Cells were treated with a series of compounds to determine the specificity of c-fos stimulation. Of the compounds tested, only PTH, prostaglandin E(2), 8-bromo-cAMP, and forskolin induced c-fos mRNA levels, indicating that this assay was specific for compounds that are known to induce cAMP and stimulate bone growth. These results indicate that a simple in vitro assay for c-fos may be a reliable method for the screening of compounds that stimulate bone growth in vivo.
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PMID:Development of an In Vitro Screening Assay for Compounds that Increase Bone Formation. 1083 33

Cytokines and proteases are secreted by fibroblasts in response to particulate wear debris, and these proteins are felt to play an important role in the development of osteolysis and implant loosening. Although metallic and polyethlyene debris have been studied extensively, little is known about the cellular responses to hydroxyapatite, despite the wide clinical use of these materials. Therefore, the effects of hydroxyapatite (HA) and hydroxyapatite/beta-tricalciumphosphate (HA/TCP) on cellular proliferation, cytokine gene expression and protein secretion, protease synthesis, and gelatinolytic activity were investigated in human fibroblasts. HA and HA/TCP particles were synthesized, and their effects were compared to the responses elicited by titanium and cobalt chromium. Sample characterization by scanning electron microscopy and Coulter Counter demonstrated that the materials had a mean particle size of less than 10 microm, and all of the particles were compared using the same concentration ranges. Aliquots of particle suspensions were added to human fibroblasts maintained in tissue culture, and dose-response and time-course experiments were performed. Effects of the particles on fibroblast proliferation were assessed, and alterations in cytokine levels were determined by specific enzyme linked immunosorbent assays (ELISA). Cytokines that were evaluated included interleukin-1 (IL-1beta), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-alpha), all of which have been demonstrated to enhance bone resorption and are associated with osteolysis and implant loosening. Gene expression was determined using Northern blot analysis with cytokine-specific probes, while secretion of the proteases collagenase and stromelysin was determined by Western blot analysis. Functional gelatinolytic assay was assessed using zymogram gels. The particles were evaluated in a concentration range from 0.000021 to 0.021 vol%. All of the particles produced increases in cellular proliferation up to 0.0021 vol%, with the largest increases being seen at 0.021 vol% with HA/TCP and titanium. At the highest concentration, both cobalt chromium and HA samples decreased cellular proliferation relative to lower doses, possibly representing cytotoxicity. Hydroxyapatite particles yielded a 30-fold increase in interleukin-6 secretion compared to unstimulated controls, which was also greater than three times the levels produced by cobalt chromium, titanium, or HA/TCP. HA particles also tripled the secretion of IL-1beta at 0.00021 vol%, and doubled TNF-alpha secretion at 0.021 vol%. Addition of conditioned media prepared by incubation of the particles in culture medium in the absence of cells did not alter the secretion of any of the cytokines. Northern blot analysis using IL-6 probes also demonstrated strong increases with HA compared to the other materials, suggesting that the action of the HA particles was at the level of transcription. Secretion of the protease collagenase was increased by all of the samples including HA when compared to unstimulated controls. Stromelysin secretion into the culture medium was decreased by cobalt chromium, but increased by titanium, HA, and HA/TCP. All of the particles including HA increased the gelatinolytic activity of the fibroblasts. These findings demonstrate that HA and HA/TCP particles are capable of stimulating the expression and secretion of cytokines and proteases that enhance bone resorption, and suggest that particulate debris from implants using these coatings may also increase osteolysis and loosening.
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PMID:Effects of hydroxyapatite particulate debris on the production of cytokines and proteases in human fibroblasts. 1151 71


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