Interleukin-17, a regulator of angiogenic factor release by synovial fibroblasts

OBJECTIVE: Angiogenesis is a process stimulated in inflamed synovium of patients with osteoarthritis (OA), and contributes to the progression of the disease. Synovial fibroblasts secrete angiogenic factors, such as vascular endothelial growth factor (VEGF), an up-regulator of angiogenesis, and this ability is increased by interleukin (IL)-1beta. The purpose of this study was to verify whether IL-17 contributes and/or synergizes with IL-1beta and tumor necrosis factor (TNF)-alpha in vessel development in articular tissues by stimulating the secretion of proangiogenic factors by synovial fibroblasts. DESIGN: We stimulated in vitro synovial fibroblasts isolated from OA, rheumatoid arthritis (RA) and fractured patients (FP) with IL-17 and IL-1beta and from OA patients with IL-17, IL-1beta and TNF-alpha. In the supernatants from the cultures, we assayed the amount of VEGF by immunoassay and other angiogenic factors (keratinocyte growth factor, KGF; hepatocyte growth factor, HGF; heparin-binding endothelial growth factor, HB-EGF; angiopoietin-2, Ang-2; platelet-derived growth factor B, PDGF-BB; thrombopoietin, TPO) by chemiluminescence; semiquantitative RT-PCR was used to state mRNA expression of nonreleased angiogenic factors (Ang-2 and PDGF-BB) and tissue inhibitors of metalloproteinase (TIMP)-1. RESULTS: IL-17, TNF-alpha and IL-1beta increased VEGF secretion by synovial fibroblasts from OA patients. IL-17 and IL-1beta also increased VEGF secretion in RA and FP. Besides, IL-17 increased KGF and HGF secretions in OA, RA and FP; in OA and RA, IL-17 also increased the HB-EGF secretion and the expression of TIMP-1 as protein and mRNA. In OA patients IL-17 had an additive effect on TNF-alpha-stimulated VEGF secretion. CONCLUSIONS: These results suggest that IL-17 is an in vitro stimulator of angiogenic factor release, both by its own action and by cooperating with TNF-alpha.     

Fibroblast expression of C-C chemokines in response to orthopaedic biomaterial particle challenge in vitro.

C-C chemokines are soluble mediators that occur in a periprosthetic granuloma and influence recruitment, localization and activation of inflammatory cells. This study tested effects of titanium and polymethylmethacrylate (PMMA) particles on expression of selected C-C chemokines in cultured human fibroblasts. The C-C chemokines analyzed included monocyte chemoattractant protein-1. 2 (MCP-1. 2), monocyte inflammatory protein-1 alpha (MIP-1 alpha), and regulated on activation, normal T-cell expressed and secreted protein (RANTES). Interleukin-1 beta (IL-1 beta) served as a known stimulator of chemokine release while interleukin-6 (IL-6) expression served as a marker for fibroblast activation. Protein and mRNA signal levels were determined by ELISA and RT-PCR, respectively. The results demonstrated that exposure of fibroblasts to titanium and PMMA particles resulted in increased release of MCP-1 in a dose- and time-dependent manner. After 24 h, titanium particles maximally upregulated MCP-1 release 7-fold while PMMA particles increased MCP-1 levels 2-fold, when compared to unchallenged fibroblasts. MCP-2, MIP-1 alpha and RANTES levels remained unchanged following exposure of fibroblasts to titanium or PMMA particles at any concentration or time point tested. However, IL-1 beta stimulated release of MCP-1, MCP-2, and RANTES, but not MIP-1 alpha from the fibroblasts. IL-1 beta, not particles, exhibited the most prominent effect on MCP-1 mRNA levels. Increased release of MCP-1 from fibroblasts exposed to titanium and PMMA particles coincided with increased release of IL-6. This study suggests that release of chemoattractant factors from fibroblasts localized in periprosthetic membranes enhances the chronic inflammatory process leading to bone resorption and implant loosening.     

Hyaluronan does not affect cytokine and chemokine expression in osteoarthritic chondrocytes and synoviocytes

OBJECTIVE: Many studies have evidenced the clinical efficacy of hyaluronan (HA) in the treatment of osteoarthritis (OA). However, human and animal studies have described proinflammatory effects of HA on cells not involved in OA. We therefore investigated whether different molecular weight HA preparations can affect proinflammatory cytokine (IL1beta and TNFalpha) or chemokine (IL8, MCP-1 and RANTES) expression in human chondrocytes and synoviocytes isolated from OA patients. DESIGN: Human chondrocytes and synoviocytes were cultured in vitro in the presence or absence of three different purified HA pharmaceutical preparations (1x10(6) Kd, 5x10(5) Kd and 6.5x10(4) Kd) and assessed for the production of proinflammatory cytokines and chemokines and their mRNA expression. RESULTS: basal conditions, both chondrocytes and synoviocytes produce only MCP-1 and IL8, along with low quantities of IL1beta and TNFalpha, but not RANTES. IL8 production was generally about 100 times higher in chondrocytes than in synoviocytes, while MCP-1 was roughly twice as high in synoviocytes than in chondrocytes. At the mRNA level, expression of IL1beta, TNFalpha, IL8, MCP-1 and RANTES did not change in the presence of the three HA preparations either in synoviocytes or in chondrocytes with respect to basal condition. None of the three different HA preparations significantly affected production of IL8 or MCP-1. CONCLUSIONS: These data demonstrate that preparations of HA of the same origin but with different MWs do not induce proinflammatory cytokines and chemokines expressed by chondrocytes and synoviocytes that are either directly or indirectly involved in OA progression.     

Proinflammatory cytokines induce NF-kappaB-dependent/NO-independent chemokine gene expression in MIN6 beta cells

BACKGROUND: Interactions between chemokines IP-10, MCP-1, and RANTES and their receptors may mediate graft rejection following islet transplantation. The mechanisms regulating chemokine gene expression in pancreatic islet cells have not been well characterized. We examined the cytokine-induced gene expression profiles for several chemokines in a transformed pancreatic beta-cell line (MIN6) cotreated with an inhibitor of nitric oxide synthase and in a mutated clone of MIN6 made to overexpress a dominant negative inhibitor of NF-kappaB (IkappaBalphaM). METHODS: MIN6 and MIN6-IkappaBalphaM (Bm) cells were cultured in mixtures of IL-1beta and TNF-alpha or IL-1beta, TNF-alpha, and IFN-gamma plus/minus the iNOS inhibitor L-NMMA. RT-PCR and RNase Protection Assay were used to measure mRNA expression for the following chemokines: IP-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES. Enzyme linked immunosorbant assay was used to measure IP-10 and MCP-1 protein release. RESULTS: Cytokine-treated MIN6 and Bm demonstrated increased expression of genes for IP-10 and MCP-1. Expression in MIN6 was first detected at 2 h of incubation and peaked at 6 h. MIN6 demonstrated a more marked increase in chemokine gene expression for both IP-10 and MCP-1 and a more marked increase in IP-10 protein release than did Bm. There was no detectable gene expression for MIP-1alpha, MIP-1beta, or RANTES from MIN6 or Bm. L-NMMA completely blocked NO production from MIN6 and Bm but had no effect on chemokine gene expression in either MIN6 or Bm. CONCLUSIONS: These results suggest that beta cells produce a complement of rejection-relevant chemokines in response to a proinflammatory stimulus and that pathways governing cytokine-induced chemokine gene expression in MIN6 are dependent on NF-kappaB but independent of NO.     

IL-17, IL-1beta and TNF-alpha stimulate VEGF production by dedifferentiated chondrocytes

OBJECTIVE: To verify the involvement of proinflammatory cytokines IL-17, IL-1beta and tumor necrosis factor alpha (TNF-alpha) in cartilage vascularization by stimulating the production of vascular endothelial growth factor (VEGF) by chondrocytes isolated from patients with osteoarthritis (OA), in comparison with patients with rheumatoid arthritis (RA) and patients with femoral or humeral neck fracture (FP). DESIGN: Chondrocytes isolated from patients with OA were maintained in monolayer culture for several passages. Chondrocyte dedifferentiation was monitored by the synthesis of cathepsin B by these cells. Chondrocytes freshly isolated at each subculture (subcultures 1-3) were stimulated with IL-17, IL-1beta or TNF-alpha. Supernatants were collected, immunoassayed for the production of VEGF and cathepsin B and assayed as the source of VEGF on the VEGF sensible ECV304 cell line. The cells were used to quantify intracellular cathepsin B enzymatic activity. RESULTS: In differentiated conditions IL-1beta and TNF-alpha, but not IL-17, can inhibit the spontaneous secretion of VEGF by human OA, RA and FP chondrocytes, and IL-17 can restore the decrease in VEGF secretion caused by TNF-alpha. IL-17, together with IL-1beta and TNF-alpha, can enhance VEGF secretion to various extents by dedifferentiated OA chondrocytes. This change in effect with respect to primary culture was observable for all cytokines at the beginning of dedifferentiation, when the production of VEGF by chondrocytes had dramatically fallen and the cathepsin B synthesis had increased. The amount of VEGF induced by cytokines on dedifferentiated chondrocytes never reached the amount of VEGF produced by differentiated chondrocytes. VEGF produced by chondrocytes stimulated the ECV304 cell line proliferation. CONCLUSIONS: These results indicate that dedifferentiated OA chondrocytes secrete VEGF after stimulation with proinflammatory cytokines. This event may be responsible for neovascularization found in OA cartilage.     

Cytokines, tumor necrosis factor-alpha and interleukin-1beta, differentially regulate apoptosis in osteoarthritis cultured human chondrocytes

OBJECTIVE: This study addresses the effects of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) on cell death in human chondrocytes. METHODS: Osteoarthritis (OA) human chondrocytes stimulated with Actinomycin-D (ActD) were used as a cellular apoptotic model. Caspase family mRNA expression and protein synthesis were analyzed by the ribonuclease protection assay and Western-blot, respectively. Cell viability and apoptosis were evaluated using the 3-[4,5-dimethylthiazol-2yl] 2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Prostaglandin E2 (PGE2) and nitric oxide (NO) were evaluated by enzyme-linked immunosorbent assay (ELISA) and the Griess method, respectively. RESULTS: TNF-alpha and IL-1beta differentially affected the pattern of caspase mRNA expression by human chondrocytes. TNF-alpha induced a gradual increase in caspase-1 and -8 mRNA levels that was not seen with IL-1beta. The time sequence of caspase-3 and -7 inductions by TNF-alpha differs from that induced by IL-1beta. Cell viability was not modified by TNF-alpha or IL-1beta in cultured chondrocytes. Then, we employed ActD as a model to facilitate cell death. Treatment with TNF-alpha and ActD (TNF-alpha/ActD) increased cell death induced by ActD (23%). Treatment with IL-1beta and ActD (IL-1beta/ActD) did not modulate ActD-induced cell death. Similarly, IL-1beta/ActD did not induce an increase in the activation of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP) cleavage observed by the incubation with TNF-alpha/ActD. These different effects were not due to bcl-2 or mcl-1 levels. Inhibition of PGE2 synthesis by indomethacin increased the cell death induced by IL-1beta/Act-D (59%). An inhibitor of caspase-8 significantly reduced only the TNF-alpha/ActD-induced cell death (58%). CONCLUSION: TNF-alpha and IL-1beta differentially regulate the apoptotic pathway in human chondrocytes. This difference is dependent on PGE2 and caspase-8 levels.     

Prevalence and consequences of musculoskeletal symptoms in symphony orchestra musicians vary by gender: a cross-sectional study

Background

Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed.

Methods

TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA.

Results

TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE₂, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA.

Conclusions

TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.     

Interleukin-17 stimulates the release of pro-inflammatory cytokines by blood monocytes in patients with IgA nephropathy

OBJECTIVE: Interleukin-17 (IL-17) is a newly discovered cytokine implicated in the regulation of inflammation. The present study was designed to explore whether IL-17 is involved in the immunoregulatory response in IgA nephropathy (IgAN). MATERIAL AND METHODS: We examined the in vitro effect of recombinant human IL-17 (rhIL-17) on pro-inflammatory cytokine release by peripheral blood monocytes (PBM) from patients with IgAN. Measurement of IL-1beta and tumor necrosis factor-alpha (TNF-alpha) was performed by means of a sandwich enzyme-linked immunosorbent assay. RESULTS: IL-1beta and TNF-alpha release were upregulated by rhIL-17 in a dose- and time-dependent fashion. Treatment of PBM from patients with IgAN with lipopolysaccharide and rhIL-17 resulted in significant activation and release of IL-1beta and TNF-alpha protein. Levels of IL-1beta and TNF-alpha were significantly higher in IgAN patients with the nephrotic syndrome (NS) than in patients without NS or in healthy subjects. We also provide information indicating that there is excessive production of IL-17 in IgAN patients. When IL-17-activated PBM were incubated in the presence of IL-10, IL-10 exerted a significant suppressive effect on IL-1beta and TNF-alpha release in vitro. CONCLUSION: IL-17 can stimulate the release of pro-inflammatory cytokines from PBM in patients with IgAN.     

Differential induction and regulation of matrix metalloproteinases in osteoarthritic tissue and fluid synovial fibroblasts

OBJECTIVES: To investigate the secretion profiles of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in synovial fluid-derived fibroblasts and to compare them with those of tissue-derived fibroblasts. METHODS: Fibroblast cultures established from synovial tissues (TSC) and fluids (FSC) of the same OA patients were stimulated with tumor necrosis factor(TNF)-alpha, interleukin(IL)-1alpha, IL-1beta, IL-6 and a combination of TNFalpha and IL-1beta. Cocultures of fibroblasts and cartilage were stimulated either with the cytokine combination or with osteoarthritic synovial fluid. Secretion of MMP-1, MMP-3, MMP-8, MMP-13, TIMP-1, and TIMP-2 was measured by enzyme-linked immunosorbent assay. Gelatin zymography and immunoblotting were performed to demonstrate enzyme activity. RESULTS: TNFalpha, IL-1alpha, and IL-1beta led to marked increases in MMP-1 and MMP-3 release (up to 4.2-fold and 547-fold, respectively) by synovial fibroblasts, whereas secretion of MMP-13 was induced by concomitant administration of TNFalpha and IL-1beta. Expression of intracellular MMP-8 was stimulated by cytokines, but adhesion of synovial fibroblasts to cartilage was required for the release. Throughout the study, significantly higher levels of secreted MMPs were observed in stimulated FSC compared to TSC cultures. Furthermore, increases in MMP secretion were not accompanied by increases in secreted TIMP-1 and TIMP-2, resulting in marked imbalances between enzyme and inhibitor levels. CONCLUSIONS: The results provide strong evidence for a significant impact of synovial-derived MMPs on cartilage destruction in OA. In this context, fibroblasts present in the synovial fluid appeared to play an outstanding role.     

Selective induction of MCP-1 in human mesangial cells by the IL-6/sIL-6R complex

Interleukin (IL) 6, an autocrine growth factor for mesangial cells, and chemokines, which are released from activated mesangial cells and induce leukocyte infiltration, play a critical role in the progression of immune system mediated renal diseases. Since the reciprocal relationship between IL-6 and chemokines in renal inflammation has been barely investigated, we have analyzed whether IL-6 (500 ng/ml), alone or in combination with the soluble form of its receptor (sIL-6R, 200 ng/ml), can induce normal human mesangial cells (NHMC) to release alpha and/or beta chemokines: MCP-1 (monocyte chemoattractant protein 1), IL-8, Rantes (regulated on activation, normal T cell expressed and secreted), and MIP-1alpha (macrophage inflammatory protein 1alpha). Whereas IL-6 or sIL-6R alone were ineffective in inducing significant chemokine release from NHMC, the simultaneous treatment with IL-6 and sIL-6R showed a significant interaction, leading to a strong synergic effect on MCP-1 synthesis and release without exerting any relevant activity on IL-8, Rantes, or MIP-1alpha. Consistently with the unresponsiveness to IL-6, mRNA and protein expression analysis of the two subunits which form the functional IL-6 receptor showed that NHMC express only the gp130 signal-transducing chain and not the subunit-specific IL-6R (gp80). These findings support an unexpected role of the IL-6 system in kidney inflammatory reactions through the selective regulation of monocyte recruitment.     

Assessment of the utility of biomarkers of osteoarthritis in the guinea pig

OBJECTIVE: To identify biochemical markers of osteoarthritis (OA) in the guinea pig, we characterized four biomarkers and 17 cytokines for age- and strain-related differences. METHODS: Two guinea pig strains were examined in this study: (1) the Hartley (OA-prone) and (2) Strain 13 (OA-resistant). Levels of synovial fluid keratan sulfate (KS) and cartilage oligomeric matrix protein (COMP), as well as levels of serum C2C, CPII, and a panel of cytokines and chemokines were quantified in both guinea pig strains. These included: IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-12p70, IL-17, G-CSF, GM-CSF, IFN-gamma, KC, MIP-1 alpha, RANTES, and TNF-alpha. RESULTS: Synovial fluid concentrations of KS and COMP increased coincident with histological OA and correlated positively with the severity of histological damage in both strains. Synovial fluid concentrations of these biomarkers were elevated in the knees of the Hartley compared to the Strain 13 animals, as early as 2 months of age. From as early as 4 months of age, the levels of serum C2C/CPII, representing the ratio of type II collagen degradation and synthesis, were elevated in the OA-prone Hartley compared with Strain 13 animals. Also, at 12 months of age, strain-related differences were apparent for 11 of the 16 cytokines and chemokines. Using multiple linear regression, serum IL-6 and TNF-alpha concentrations were each strongly associated with strain, weight, and their interaction (r2 = 0.80, P = 0.0002 for IL-6; r2 = 0.55, P = 0.02 for TNF-alpha). CONCLUSIONS: Biomarkers derived from synovial fluid are reflective of histological severity in the spontaneous model of OA in the guinea pig. The synovial fluid biomarker profiles indicated accelerated cartilage matrix turnover in the Hartley strain as early as 2 months of age, prior to evidence of histological damage. The Hartley strain also exemplified an imbalance in type II collagen metabolism and a serum cytokine/chemokine profile indicative of a pro-inflammatory state. These findings elucidate additional disease-related features in the guinea pig that have relevance to OA in humans.     

Interleukin-17: A new bone acting cytokine in vitro.

Interleukin-17 (IL-17) is a recently cloned cytokine that is exclusively produced by activated T cells, but its receptor has been found on several cells and tissues. Like other proinflammatory cytokines produced by activated T cells, IL-17 may affect osteoclastic resorption and thereby mediate bone destruction accompanying some inflammatory diseases. In the present study, we investigated whether osteogenic cells possess the receptor for IL-17 (IL-17R) and whether IL-17 affects osteoclastic resorption. We found that IL-17R mRNA is expressed both in mouse MC3T3-E1 osteoblastic cells and fetal mouse long bones, suggesting that osteogenic cells may be responsive to IL-17. In fetal mouse long bones, IL-17 had no effect on basal and IL-1beta-stimulated osteoclastic bone resorption, but when given together with tumor necrosis factor-alpha (TNF-alpha) it increased bone resorption dose dependently in serum-free conditions. In addition, IL-17 increased TNF-alpha-induced IL-1alpha, IL-1beta, and IL-6 mRNA expression in fetal mouse metatarsals and IL-1alpha and IL-6 mRNA expression in MC3T3-E1 cells. In conclusion, IL-17R mRNA was expressed by mouse osteoblastic cells and fetal mouse long bones, and IL-17 in combination with TNF-alpha, but not IL-1beta, increased osteoclastic resorption in vitro. IL-17 may therefore affect bone metabolism in pathological conditions characterized by the presence of activated T cells and TNF-alpha production such as rheumatoid arthritis and loosening of bone implants.     

Osteoblasts from the sclerotic subchondral bone downregulate aggrecan but upregulate metalloproteinases expression by chondrocytes. This effect is mimicked by interleukin-6, -1beta and oncostatin M pre-treated non-sclerotic osteoblasts

OBJECTIVE: To determine the effects of osteoarthritic (OA) subchondral osteoblasts on the metabolism of human OA chondrocytes in alginate beads. METHODS: Human chondrocytes were isolated from OA cartilage and cultured in alginate beads for 4 days in the absence or in the presence of osteoblasts isolated from non-sclerotic (N) or sclerotic (SC) zones of human OA subchondral bone in monolayer (co-culture system). Before co-culture, osteoblasts were incubated for 72 h with or without 1.7ng/ml interleukin (IL)-1beta, 100 ng/ml IL-6 with its soluble receptor (50 ng/ml) or 10 ng/ml oncostatin M (OSM). Aggrecan (AGG) and matrix metalloproteases (MMP)-3 and -13 mRNA levels in chondrocytes were quantified by real-time polymerase chain reaction. AGG production was assayed by a specific enzyme amplified sensitivity immunoassay. RESULTS: SC, but not N, osteoblasts induced a significant inhibition of AGG production and AGG gene expression by human OA chondrocytes in alginate beads, and significantly increased MMP-3 and MMP-13 gene expression by chondrocytes. When they were pre-incubated with IL-1beta, IL-6 or OSM, N osteoblasts inhibited AGG synthesis and increased MMP-3 and -13 gene expression by chondrocytes in alginate beads in a same order of magnitude as SC osteoblasts. CONCLUSIONS: These results demonstrate that SC OA subchondral osteoblasts could contribute to cartilage degradation by stimulating chondrocytes to produce more MMP and also by inhibiting AGG synthesis.     

Monocyte chemoattractant protein-1 production by human detrusor smooth muscle cells

PURPOSE: Most recently attention has turned to the secretory properties of smooth muscle cells. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemokine that causes mast cells recruitment and provokes mast cells activation in vitro. We investigated whether MCP-1 is produced by human detrusor smooth muscle cells (HDSMCs) cultured under inflammatory conditions. MATERIALS AND METHODS: Using an explantation technique HDSMCs were isolated and short-term cultured. HDSMCs were incubated at 37C for 24 hours with the proinflammatory mediators interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF-alpha), lipopolysaccharide, histamine, leukotriene D4 and prostaglandin E2. The level of MCP-1 in cell supernatants were measured by enzyme linked immunoassay. RESULTS: There was basal secretion of MCP-1 in unstimulated cultures. Following 24-hour incubation with IL-1 beta or TNF-alpha (1 pg/ml to 100 ng/ml) the level of MCP-1 increased in a dose dependent manner. IL-1 beta was more potent at inducing MCP-1 release in 8 of 10 experiments. Lipopolysaccharide (10 microg/ml), histamine (100 microM), leukotriene D4 (50 nM), prostaglandin E2 (1 microM) and KCl (30 to 100 mM) failed to induce MCP-1 production. When IL-1 beta (10 ng/ml) and TNF-alpha (10 ng/ml) were given in combination, a highly synergistic effect on MCP-1 production was observed. CONCLUSIONS: To our knowledge this study shows for the first time that human detrusor smooth muscle cells cultivated under inflammatory conditions produce significant amounts of MCP-1. In addition to contractile function, HDSMCs have synthesis and secretory potential with the release of MCP-1. Thus, MCP-1 may contribute to the local inflammatory process by producing proinflammatory mediators. The release of cytokines and chemokines by human detrusor muscle even in small amounts may have important functional consequences.     

Receptor for advanced glycation end products on human synovial fibroblasts: role in the pathogenesis of dialysis-related amyloidosis

An important component of amyloid fibrils in dialysis-related amyloidosis (DRA) is beta(2)-microglobulin (beta(2)m) modified with advanced glycation end products (AGE). The amyloid deposits are located principally in joint structures, with adjacent chronic inflammatory reaction characterized by monocyte infiltration. This study examined the interaction of AGE-beta(2)m with human synovial fibroblasts and investigated the proinflammatory effects of that interaction. It was demonstrated that human synovial fibroblasts constitutively expressed the receptor for AGE (RAGE). RAGE expression was detected mainly in synovial intima and was upregulated in DRA synovium. (125)I-AGE-beta(2)m bound to immobilized human synovial fibroblasts in a specific, dose-dependent manner (K(d) of approximately 138.0 nM), and binding was inhibited by anti-RAGE IgG. Incubation of human synovial fibroblasts with AGE-beta(2)m induced degradation of this AGE-modified protein, as well as increased monocyte chemoattractant protein-1 (MCP-1) mRNA and protein expression. The amount of MCP-1 produced by AGE-beta(2)m-stimulated human synovial fibroblasts was sufficient to induce the chemotaxis of monocytes. MCP-1 synthesis resulted from engagement of RAGE, because the increase in MCP-1 synthesis was attenuated by preincubation of human synovial fibroblasts with anti-RAGE IgG. These data provide evidence of RAGE-mediated perturbation of human synoviocytes, which may be involved in the pathogenesis of inflammatory processes associated with DRA.     

Organ-specific regulation of pro-inflammatory molecules in heart, lung, and kidney following brain death

BACKGROUND: Nonspecific inflammatory events following brain death may increase the intensity of the immunological host response. The present study investigated the course of pro-inflammatory molecules in heart, lung, kidney, and plasma after brain death induction. MATERIALS AND METHODS: Brain death was induced in five pigs by inflation of an intracranial Foley catheter and five pigs were sham-operated as controls. Each experiment was terminated 6 h after brain death/sham operation and the organs were harvested. We measured the mRNA and protein levels for TNF-alpha, IL-1beta, and IL-6 in heart, lung, kidney, and plasma. Additionally, the mRNA expression for IL-6R, ICAM-1, MCP-1, and TGF-beta was determined in each organ. RESULTS: After 6 h, the plasma cytokine levels were higher in the brain-dead animals than in the sham-operated. In heart, lung, and kidney there was an increase in IL-6 and IL-1beta following brain death, while TNF-alpha was up-regulated in lung only (P < 0.05). MCP-1 and TGF-beta were significantly higher in heart and lung and IL-6R increased in heart after brain death (P < 0.05). CONCLUSIONS: Brain death was associated with non-uniform cytokine expression patterns in the investigated organs. These expression patterns may cause variable pro-inflammatory priming resulting in different degrees of damage and explain the organ-specific variation in outcomes after transplantations.