beta(2)-Microglobulin modified with advanced glycation end products delays monocyte apoptosis

BACKGROUND: A local inflammatory reaction to beta(2)-microglobulin (beta(2)m) amyloid deposits by monocytes/macrophages is a characteristic histologic feature of dialysis-related amyloidosis (DRA). Since beta(2)m modified with advanced glycation end products (AGE-beta(2)m) is a major constituent of amyloid in DRA, we tested the hypothesis that AGE-beta(2)m affects apoptosis and phenotype of human monocytes. METHODS: Human peripheral blood monocytes were incubated with or without in vitro-derived AGE-beta(2)m, and their viability, extent of apoptosis, morphology, and function examined over the subsequent four days. RESULTS: AGE-modified but not unmodified beta(2)m significantly delayed spontaneous apoptosis of human peripheral blood monocytes in adherent and nonadherent cultures. The effect of AGE-beta(2)m on monocytes apoptosis was time- and dose-dependent and was attenuated by a blocking antibody directed against the human AGE receptor (RAGE). There was no difference in effect between AGE-beta(2)m and that of AGE-modified human serum albumin. Culture of monocytes with AGE-beta(2)m did not alter membrane expression of Fas or Fas ligand. Monocytes cultured with AGE-beta(2)m underwent substantial changes in morphology similar to those observed when monocytes differentiate into macrophages. The cultured cells increased in size and vacuolization, and their content of beta-glucuronidase and acid phosphatase increased by 5- to 10-fold at day 4. Expression of the monocyte--macrophage membrane antigens HLA-DR, CD11b, and CD11c also increased at day 4. Although exhibiting phenotypic characteristics of macrophages, monocytes cultured with AGE-beta(2)m functioned differently than macrophages cultured with serum. Superoxide production in response to phorbol myristic acetate was maintained in monocytes cultured with AGE-beta(2)m, but declined with time in cells cultured with serum. Constitutive synthesis of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE2) increased in monocytes cultured for four to six days with AGE-beta(2)m. CONCLUSIONS: These findings support a novel role for AGE-modified proteins such as AGE-beta(2)m that may contribute to the development of a local inflammatory response, with predominant accumulation of monocytes/macrophages, in DRA.     

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.     

Proximal tubule cells stimulated by lipopolysaccharide inhibit macrophage activation

BACKGROUND: Tubule cells can produce a variety of cytokines, extracellular matrix (ECM) components, and adhesion molecules in vitro and in vivo. It is generally assumed that stimulated tubule cells are proinflammatory and at least partially responsible for interstitial inflammation. However, the overall effect of tubular cells on interstitial cells is unknown. In this study, pro- and anti-inflammatory cytokine production and net effects on macrophages of tubule cells activated by lipopolysaccharide (LPS) were examined. METHODS: Tubule cells stimulated with LPS expressed tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-12, monocyte chemoattractant protein-1 (MCP-1), IL-10, and transforming growth factor-beta (TGF-beta). Conditioned media were collected from confluent monolayers of rat tubule cells stimulated, or not, by LPS for 4 and 18 hours, respectively. Macrophages were cultured with conditioned media and/or LPS (0.5 microg/mL) for 18 hours. RESULTS: TNF-alpha and IL-lbeta mRNA of macrophages stimulated by LPS increased more than fivefold when cultured with control conditioned media from unstimulated tubule cells. Surprisingly, TNF-alpha and IL-lbeta levels of macrophages stimulated by LPS were not increased when cultured with conditioned media from activated tubule cells. Neutralizing antibodies to IL-10 and TGF-beta were used to define the inhibitory component(s) in conditioned medium. Anti-IL-10, but not anti-TGF-beta, abolished partially the inhibitory effects of conditioned media on macrophages. CONCLUSION: Tubule cells produce both pro- and anti-inflammatory cytokines and the net effect, partially explained by IL-10, of tubule cells activated with LPS is to inhibit activity of macrophages. Thus, the net effect of activated tubule cells on interstitial pathology may in certain circumstances, be anti- rather than pro-inflammatory.     

Differential regulation of monocyte/macrophage cytokine production by pressure

BACKGROUND: Cytokine production by macrophages is essential for the inflammatory response. Normal human interstitial tissue pressure is 20 to 30 mm Hg, but generally decreases in acute inflammation. METHODS: We compared the effect of 20 mm Hg increased pressure (approximating normal interstitial tissue pressure) with that of ambient pressure (resembling pressure in inflamed tissues) on tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production by undifferentiated (monocytic) and PMA (phorbol 12-, myristate 13-acetate)-differentiated (macrophage-like) THP-1 cells with or without lipopolysaccharide (LPS) (10 ng/mL). RESULTS: Pressure stimulated spontaneous macrophage TNF-alpha secretion (30.5 +/- 6.3 vs. 49.1 +/- 2.8 pg/mL, P <.02), but not monocyte TNF-alpha secretion. Pressure did not stimulate IL-1beta release. As expected, LPS increased basal cytokine release. After LPS stimulation, pressure still tended to stimulate macrophage TNF-alpha, but inhibited monocyte TNF-alpha secretion (P <.05). In contrast, pressure inhibited IL-1beta release by both LPS-treated monocytes (986 +/- 134 vs. 595 +/- 226 pg/mL, P <.02) and macrophages (3,112 +/- 229 vs. 979 +/- 61 pg/mL, P <.01). CONCLUSIONS: Extracellular pressure may regulate TNF-alpha and IL-1beta secretion differentially by monocytes and macrophages.     

TNF Alpha−308 Genotype and Renin–Angiotensin System in Hemodialysis Patients: An Effect on Inflammatory Cytokine Levels?

BACKGROUND: Renin-angiotensin system (RAS) was suggested to modulate inflammatory cytokine production. Angiotensin II was consistently shown to increase production of tumor necrosis factor alpha (TNF-alpha). However, inflammatory cytokines and RAS were modulated by genetic polymorphisms such as TNF-alpha-308 G > A and angiotensin-converting enzyme (ACE) I/D gene polymorphisms. The aim of this study was to investigate the effects of ACE and TNF-alpha genotypes on inflammatory cytokines in hemodialysis (HD) patients. METHODS: ACE I/D and TNF-alpha-308 G > A genotypes, pre- and postdialysis plasma renin activity (PRA), serum ACE, interleukin-1 beta (IL-1beta), and TNF-alpha levels were determined in 22 HD patients. RESULTS: Predialysis serum ACE activity is correlated with TNF-alpha (r = 0.63; P = 0.01), and PRA was correlated with IL-1beta levels (r = 0.49; P = 0.02). Pre/postdialysis IL-1beta and TNF-alpha were similar in DD and II/ID ACE genotypes. Predialysis TNF-alpha and IL-1beta (32.4 +/- 5; 35.1 +/- 4.2 vs. 28.1 +/- 3.7; 26.5 +/- 6.2 pg/mL; P < 0.05) and postdialysis TNF-alpha levels (30.4 +/- 1.4 vs. 28.4 +/- 0.82 pg/mL; P < 0.05) were significantly higher in TNF1/2 than TNF1/1 patients. CONCLUSION: ACE and TNF-alpha-308 G > A (1/2) gene polymorphisms may contribute to modulation of proinflammatory cytokine production and hence chronic inflammation in HD patients.     

Whole blood production of monocytic cytokines (IL-1beta, IL-6, TNF-alpha, sIL-6R, IL-1Ra) in haemodialysed patients.

BACKGROUND: The production of monocytic cytokines by isolated mononuclear cells after stimulation by phytohaemagglutinin (PHA) and lipopolysaccharide (LPS) is generally increased in haemodialysed (HD) patients. We performed whole blood (WB) cultures to evaluate cytokine production by blood cells inside their complex cellular and humoral network. METHODS: Diluted whole blood from HD patients (collected before dialysis) and controls was cultured alone with PHA (2.5 microg/ml) or LPS (1 and 3 microg/ml). Supernatants were collected after 24 and 48 h of culture, and concentrations of IL-1 beta, IL-6, TNF-alpha, sIL-6R and IL-1Ra were determined by ELISA. RESULTS: The low spontaneous production of IL-1beta, IL-6 and TNF-alpha in both patients and controls was not significantly modified by PHA. The lower dose of LPS (1 microg/ml) induced a significant but lower increase in production of IL-1beta, IL-6 and TNF-alpha in patients than in controls. In contrast, while it did not further increase their production in controls, the higher concentration of LPS (3 microg/ml) still increased their production in patients to the same level than in controls. The plasma concentrations of sIL-6R were higher in patients than in controls. In both groups, the sIL-6R concentration did not vary during the culture period whether the cells were stimulated or not with LPS or PHA. This suggests that the increased plasma levels of sIL-6R were not produced by blood cells. Despite a similar significant LPS and PHA induced production of IL-1Ra, the IL-1Ra/IL-1beta ratio was always higher in patients than in controls. CONCLUSION: Monocytes from HD patients in WB cultures are hyporesponsive to PHA and LPS for their IL-1beta, TNFalpha and IL-6 production in contrast to isolated monocytes that demonstrate signs of activation. If it reflects the in vivo situation it could partly explain the immune defect in uraemic and haemodialysed patients. Higher sIL-6R/IL-6 and IL-1Ra/IL-1beta ratios could also participate to the complex immune disturbances of HD patients by reducing the biological activity of two cytokines playing a major role in the immune and inflammatory network.     

An association between inflammatory state and left ventricular hypertrophy in hemodialysis patients

This study was performed to investigate the potential relationship between left ventricular hypertrophy (LVH) and proinflammatory cytokines in hemodialysis (HD) patients and the effect of HD on cytokine production. Serum interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) measurements and echocardiographic studies were performed in 35 stable HD patients. A variety of probable risk factors for LVH including age, HD duration, blood pressure (BP), body mass index, lipid profile, hemoglobin, albumin, parathormone and homocysteine levels were also investigated. Additionally, the effect of HD procedure on cytokine levels was evaluated. Predialysis serum levels of IL-1beta, IL-6, TNF-alpha, and homocysteine in HD patients were compared with 12 healthy subjects. Left ventricular hypertrophy was demonstrated in 20 (57%) of HD patients by echocardiography. Left ventricular mass index (LVMI) was correlated positively with systolic BP (r=0.556, p=0.001), diastolic BP (r=0.474, p=0.004), and serum levels of TNF-alpha (r=0.446, p=0.009). Multiple regression analysis showed that systolic BP and TNF-alpha levels were significant independent predictors of LVH. No relationship was observed between LVH and other parameters. The mean predialysis serum level of IL-6 was significantly higher in HD patients compared to healthy controls (15.7 +/- 8.7 vs. 7.3 +/- 0.7 pg/ mL, p=0.001). Predialysis serum levels of TNF-alpha in HD patients were higher when compared to healthy subjects, but the difference was not statistically significant (8.3 +/- 3 vs. 7 +/- 1.45 pg/mL, respectively, p>0.05). However, serum levels of IL-6 and TNF-alpha significantly elevated after HD, when compared to predialysis levels (from 15.7 +/- 8.7 to 17.8 +/- 9.5 pg/mL, p=0.001 and from 8.3 +/- 3.0 to 9.9 +/- 3.5 pg/mL p=0.004, respectively). As a conclusion, in addition to BP, proinflammatory cytokines, TNF-alpha in particular, seem to be associated with LVH in ESRD patients.     

Melanoma inhibits macrophage activation by suppressing toll-like receptor 4 signaling

BACKGROUND: Activated macrophages defend against tumors by secreting cytokines to recruit secondary immune cells, presenting antigen to T cells, and by direct tumor cytotoxicity. Peritoneal macrophages harvested from melanoma-bearing mice are less cytotoxic to melanoma cells, and produce less superoxide, nitric oxide, and tumor necrosis factor-alpha (TNF-alpha) than those from nontumor-bearing mice. Similar impairment of macrophage activation occurs in vitro using media harvested from cultured melanoma cells. Stimulation of Toll-like receptor 4 (TLR-4) activates macrophages and results in the release of TNF-alpha. We hypothesized that melanoma inhibits macrophage activation by suppressing TLR-4 signaling. STUDY DESIGN: Melanoma conditioned media (MCM) was generated from B16 melanoma cells. Peritoneal macrophages from TLR-4 competent or TLR-4 incompetent mice were exposed to control or MCM for 24 hours; then stimulated with lipopolysaccharide. TNF-alpha secretion, TNF-alpha mRNA production, nuclear factor-kappaB (NF-kappaB) activation, and TLR-4 surface expression were measured. RESULTS: Peritoneal macrophages exposed to MCM produced considerably less TNF-alpha in response to stimulus than controls (691 pg/mL versus 2,066 pg/mL, p < 0.001). TNF-alpha production by TLR-4 incompetent macrophages was not affected by MCM (454 pg/mL versus 480 pg/mL). Stimulated TNF-alpha mRNA and activated NF-kappaB were decreased in MCM treated C57BL/6 macrophages (by 38% and 33%, respectively). TLR-4 surface expression, however, was not decreased by exposure to MCM. CONCLUSIONS: Melanoma inhibits macrophage activation by suppressing TLR-4 signaling downstream of the TLR-4 receptor.     

Lack of plasma interleukin-1 beta or tumor necrosis factor-alpha elevation during unfavorable hemodialysis conditions.

Plasma interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) were determined by ELISA in 17 healthy controls, 23 HD patients, 10 continuous ambulatory peritoneal dialysis patients, and 15 chronic renal failure patients, as well as in 2 HD patients experiencing pyrogenic reactions. Another group of 10 chronic HD patients were dialyzed for 2.5 h, 5 with first-use Cuprophan membranes and 5 with first-use high-flux cellulose triacetate membranes. The mean bacterial and endotoxin concentrations of the dialysate used for HD treatments during the study period were 18,440 +/- 530 CFU/mL (mean +/- SEM) and 976 +/- 205 pg/mL, respectively. Blood specimens were obtained intradialysis and postdialysis for cytokine assay and were incubated to augment cytokine production. There was no difference in plasma IL-1 beta or TNF-alpha concentrations among the healthy controls, continuous ambulatory peritoneal dialysis patients, chronic renal failure patients, or HD patients. Neither cytokine increased significantly during or after HD. Two patients experiencing pyrogenic reactions had plasma TNF-alpha concentrations of 537 and 413 pg/mL, compared with matched controls of 6 and 0 pg/mL. Il-1 beta concentration did not differ from controls. We conclude that: (1) plasma IL-1 beta and TNF-alpha are not chronically elevated in chronic renal failure, continuous ambulatory peritoneal dialysis, or HD patients; (2) HD with new Cuprophan or cellulose triacetate membranes and high concentrations of dialysate endotoxin and bacteria does not cause elevation of circulating IL-1 beta or TNF-alpha; and (3) pyrogenic reactions might be mediated by TNF-alpha.     

beta2-microglobulin induces MMP-1 but not TIMP-1 expression in human synovial fibroblasts

BACKGROUND: beta2-Microglobulin (beta2m) amyloidosis is a destructive articular disease that causes significant morbidity in patients undergoing hemodialysis. The amyloid deposits contain beta2m, some of which is altered with advanced glycation end products (AGE-beta2m). The deposits are located principally in joint structures, with adjacent degradation of cartilage and bone. We hypothesized that one of the mechanisms by which beta2m induces joint destruction is to induce the release of matrix metalloproteinase-1 (MMP-1), but not tissue inhibitor of metalloproteinase-1 (TIMP-1), from synovial fibroblasts. METHODS: To test this hypothesis and determine the role of AGE-beta2m, we incubated human osteoarthritic synovial fibroblasts in the presence and absence of beta2m and AGE-beta2m and measured the release of interstitial collagenase (MMP-1) and/or TIMP-1 by enzyme-linked immunosorbent assay and Northern blot analysis. RESULTS: beta2m and AGE-beta2m at 10 and 25 microg/mL induced the release of MMP-1 from human osteoarthritic synovial fibroblasts at 24 hours. In contrast, there was no increased release of TIMP-1, leading to an increase in the MMP-1/TIMP-1 ratio indicative of uncontrolled collagenolysis. A similar dose response was observed at 48 hours, except that AGE-beta2m had no effect over control cultures. MMP-1 mRNA expression by Northern blot analysis paralleled these findings. The source of the fibroblasts did not alter the results. Finally, we demonstrated that doxycycline, a treatment for arthritis, can inhibit the release of MMP-1 from synovial fibroblasts incubated with beta2m. CONCLUSION: beta2m, at physiologically relevant concentrations, induces the release of MMP-1 without concomitant release of TIMP-1 from human synovial fibroblasts, leading to uncontrolled collagenolysis. The alteration of beta2m with AGE did not alter this effect at 24 hours, but blocked the effect at 48 hours. These findings may account for the tissue destruction seen in beta2m amyloidosis.     

Ganciclovir pharmacokinetics and cytokine dynamics in renal transplant recipients with cytomegalovirus infection

Ganciclovir is considered to be the first-line treatment for cytomegalovirus (CMV) in renal transplant recipients. This infection is also associated with elevations of specific plasma cytokines post-transplantation. To investigate daily cytokine response to therapy and ganciclovir pharmacokinetics, 4 transplant recipients (3 males, 1 female) with stable renal allograft function diagnosed with CMV infection were enrolled less than 4 months post-transplant. A creatinine clearance (ClCr) was generated by the Cockroft-Gault (C-G) equation (range: 42.3-68.5 mL/min) to determine ganciclovir dosing. Blood samples were collected for ganciclovir and cytokine [including interleukin (IL)-1beta, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, TNF-alpha, GM-CSF, and interferon (IFN)-gamma analyses after 7 d of intravenous (i.v.) ganciclovir (dosage range: 165-400 mg daily) therapy and again after 7 d of oral (p.o.) ganciclovir (dosage range: 1000 mg, 2-3 times daily) therapy. Pharmacokinetic ganciclovir was described with a two-compartment model. Total clearance of ganciclovir was consistently greater than ClCr, suggesting tubular secretion. Peak concentrations for i.v. ganciclovir averaged 8.39+/-1.87 microg/mL with minimum concentrations of 0.48+/-0.35 microg/mL. Plasma concentrations were lower but more sustained during a p.o. dosing interval (max=2.12+/-0.58 microg/mL, min=1.15+/-0.34 microg/mL). IL-6, IL-8, IL-10, and TNF-alpha were detectable at multiple times during the study periods while the remainder of the cytokines were only intermittently detectable. Average concentrations (i.v. versus p.o. study period) for TNF-alpha were 40.1+/-17.5 versus 22.1+/-11.2 pg/mL, for IL-8 were 17.1+/-15.6 versus 4.12+/-2.59 pg/mL, and for IL-10 were 7.39+/-5.54 versus 2.64+/-1.06 pg/mL. Concentrations were similar for IL-6 during both studies (9.39+/-5.42 versus 14.7+/-14.8 pg/mL). TNF-alpha, IL-8, and IFN-gamma appeared to correlate with CMV antigenemia. Further investigation of ganciclovir disposition and changes in plasma cytokines in renal transplant recipients during CMV infection may provide insight into variable antiviral responses in renal transplant recipients.     

Cellular uptake of beta2M and AGE-beta2M in synovial fibroblasts and macrophages.

BACKGROUND: Beta-2-microglobulin (beta(2)M) amyloidosis is a destructive articular disease affecting dialysis patients. The amyloid deposits contain both beta(2)M and beta(2)M altered with advanced glycation end products (AGE-beta(2)M). We have shown that beta(2)M increases the expression of matrix metalloproteinase-1, vascular cell adhesion molecule-1 and cyclooxygenase-2 in human synovial fibroblasts, while the effect of AGE-beta(2)M in this model is markedly reduced. Conversely, in human monocyte/macrophages, AGE-beta(2)M stimulates cytokine release whereas beta(2)M is less potent. METHODS: To understand why the two forms of beta(2)M produce variable responses in different cells, AGE-beta(2)M was labelled with the fluorochrome Cy5, and beta(2)M was labelled with the fluorochrome Texas Red (TR) and the uptake of 50 microg/ml of each was examined through live cell imaging at different time points using confocal microscopy. RESULTS: In human synovial fibroblasts, the AGE-beta(2)M-Cy5 could be seen in endosome-like structures inside cells by 45 min. After 3.5 h the distribution of endosome-like structures had become perinuclear in nature and the concentration of AGE-beta(2)M-Cy5 within these structures had increased. When a 20-fold excess of AGE-BSA was added to the synovial fibroblasts with the AGE-beta(2)M-Cy5, the endosome-like particles were not seen, suggesting competitive inhibition of uptake through an AGE-receptor. In contrast, beta(2)M-TR progressively concentrated along the surface of synovial fibroblasts with minimal cellular uptake indicated by faint endosome-like structures seen only after 8 h. Interestingly, in a different model, human and mouse monocyte/macrophages, the AGE-beta(2)M-Cy5 and beta(2)M-TR had similar patterns of distribution and kinetics of uptake. CONCLUSION: Our results suggest that beta(2)M and AGE-beta(2)M are endocytosed via different mechanisms in human synovial fibroblasts and monocytes/macrophages. These results may offer a potential explanation of differences observed in cell culture experiments.     

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.     

Ketamine suppresses proinflammatory cytokine production in human whole blood in vitro.

The production of proinflammatory cytokines, such as tumor necrosis factor (TNF) a, interleukin (IL)-6, and IL-8, increases in patients with sepsis; marked production causes organ failure and septic shock. We previously reported that ketamine suppressed lipopolysaccharide (LPS)-induced TNF-alpha production in mice. However, there are no reports on the effect of ketamine on cytokine production in human whole blood. Therefore, in this study, we investigated the efficacy of ketamine on LPS-induced TNF-alpha, IL-6, and IL-8 production and recombinant human (rh) TNF-a-induced IL-6 and IL-8 production in human whole blood. After adding different doses of ketamine to whole blood, the blood was stimulated with LPS or rhTNF. After incubation, the plasma TNF-alpha activity and IL-6 and IL-8 concentrations were measured using the L929 cell cytotoxic assay or an enzyme-linked immunoassay. Ketamine significantly suppressed LPS-induced TNF-alpha production at concentrations >20 microg/mL. At concentrations >100 microg/mL, ketamine also significantly suppressed both LPS-induced and rhTNF-induced IL-6 and IL-8 production. In this study, we demonstrated that ketamine directly inhibits the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-8 in human whole blood. IMPLICATIONS: We found that ketamine suppressed lipopolysaccharide-induced tumor necrosis factor alpha, interleukin (IL)-6, and IL-8 production and recombinant human tumor necrosis factor-induced IL-6 and IL-8 production in human whole blood. Ketamine directly suppresses proinflammatory cytokine production.     

TNF-alpha and IL-1beta-mediated regulation of MMP-9 and TIMP-1 in renal proximal tubular cells

BACKGROUND: Tubulointerstitial fibrosis is a morphologic hallmark of chronic kidney disease and is a key factor in the prediction of progression to end-stage renal failure. Disruption of tubular basement membrane and interstitial extracellular matrix (ECM) via cytokine-induced alterations in matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) may be an important mechanism in this process. The presence of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) and their effects on proximal tubular cells may be critical in this process. METHODS: Human proximal tubular cells were cultured in hormonally defined medium. Cells at 80% confluency were exposed to TNF-alpha (0.1 to 100 ng/mL) or IL-1beta (0.1 to 100 ng/mL) or a combination of both for 48 hours. Activity and expression of MMP-9 was examined by gelatin zymography and Western blot analysis. TIMP-1 expression was analyzed by Western blotting. Signaling through cytokine receptors, protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways was investigated. RESULTS: TNF-alpha but not IL-1beta resulted in a dose-dependent increase in the latent form of MMP-9. TIMP-1 was decreased by treatment with either TNF-alpha or IL-1beta. Cotreatment with IL-1beta abolished the induction of MMP-9 but augmented the inhibition of TIMP-1 in the presence of TNF-alpha. Inhibition of PKC provided evidence of the importance of this pathway in mediating the cytokine-induced suppression of TIMP-1 in human kidney (HK-2) cells. Activation of the extracellular signal-regulated protein kinase (ERK1/2) MAPK mediated the up-regulation of MMP-9 by TNF-alpha whereas p38 was found to be involved in the IL-1beta-mediated inhibition of TNF-alpha-stimulated MMP-9. CONCLUSION: The differential effects of TNF-alpha and IL-1beta on proximal tubular MMP-9 and TIMP-1 expression are mediated through the TNF-RI, the IL-1-RI and the different signaling pathways of PKC, ERK1/2, and p38 MAPK. These findings may provide new insights into the role of proinflammatory cytokines TNF-alpha and IL-1beta in the development and possible therapeutic intervention in tubulointerstitial fibrosis.     

Bupivacaine's action on the carrageenan-induced inflammatory response in mice: cytokine production by leukocytes after ex-vivo stimulation

We aimed to study the effect of bupivacaine on the systemic response elicited by intraplantar injection of carrageenan. To that purpose, we studied the effects of carrageenan, bupivacaine, or both on the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 by whole blood cultured in the presence of lipopolysaccharide (LPS) and of heat-killed Staphylococcus Aureus Cowan (SAC). Mice received a hindpaw injection of carrageenan with or without encapsulated IM bupivacaine given contralaterally. Whole blood was sampled 15 h later and cultured for 24 h with LPS or SAC. The amounts of TNF-alpha, IL-1beta, and IL-10 in the supernatants were measured. In the presence of LPS or SAC, proinflammatory cytokine (TNF-alpha and IL-1beta) production was increased after carrageenan. Bupivacaine prevented this inflammatory response: 992 +/- 102 versus 2146 +/- 338 versus 919 +/- 116 pg/mL for TNF-alpha (bupivacaine + carrageenan versus carrageenan versus control after LPS stimulation). This effect of bupivacaine was less after SAC stimulation. Moreover, IL-10 was not involved in the inhibition of proinflammatory cytokine production observed after treatment by bupivacaine alone. These experiments show that carrageenan-induced hindpaw inflammation modifies the blood cell reactivity to LPS and SAC and that bupivacaine regulates the systemic response elicited by carrageenan. Furthermore, IL-10 does not seem to be a factor of the antiinflammatory response induced by bupivacaine. The precise mechanism underlying this effect of bupivacaine remains to be clarified.     

The effect of epidural clonidine on perioperative cytokine response, postoperative pain, and bowel function in patients undergoing colorectal surgery

The postoperative period is associated with an increased production of cytokines, which augment pain sensitivity. We investigated the hypothesis that epidural clonidine premedication and postoperative patient-controlled epidural analgesia (PCEA) including clonidine would decrease the release of proinflammatory (interleukin (IL)-6, IL-1beta, IL-8, and tumor necrosis factor (TNF)-alpha) and antiinflammatory (IL-1 receptor antagonist (RA)) cytokines in patients who underwent elective colorectal surgery and that they would provide better postoperative analgesia. Forty patients were randomly assigned to 1 of 2 groups of 20 each: the control group received normal saline 10 mL, whereas the clonidine group received epidural clonidine 150 microg diluted with 9 mL of normal saline 30 min before surgery. Venous blood samples for cytokine levels were obtained before induction, at the end of surgery, and after surgery at 12 and 24 h. After surgery, the clonidine group patients received PCEA with morphine (0.1 mg/mL) and clonidine (1.5 microg/mL) in 0.2% ropivacaine 100 mL, whereas control group patients received only PCEA morphine and ropivacaine. Patients in the clonidine group exhibited longer PCEA trigger times, lower pain scores at rest and while coughing, less morphine consumption, and a faster return of bowel function throughout the 72-h postoperative observation period, compared with patients in the control group. For patients in the clonidine group, production of IL-1RA, IL-6, and IL-8 was significantly less increased at the end of the surgical procedure and at 12 and 24 h after surgery. However, the concentrations of IL-1beta and TNF-alpha were not significantly increased.     

Modulation of IL-1beta,IL-6, TNF-alpha and PGE(2) by pharmacological agents in explants of membranes from failed total hip replacement

Introduction and goal Proinflammatory cytokines and prostaglandin E(2) (PGE(2)) play an important role in the pathophysiology of osteolysis and implant loosening. The aim of this study was to evaluate the role of pharmacological agents in the inhibition of Interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) and PGE(2) in explants of interface membranes from failed total hip replacements (fTHR).Material and methods Membranes from fTHR were retrieved (N=20) and explants were incubated for 72h in the absence or presence of tenidap at three different concentrations (5, 20 or 50 microg/ml) or diclofenac (125 microg/l). IL-1beta, IL-6, TNF-alpha, and PGE(2) levels were measured in the culture medium using ELISA Capture or EIA kits. Statistical analysis was done using the Mann-Whitney U-test.Results A statistically significant inhibition in IL-1beta synthesis was found at tenidap concentrations of 20 microg/ml (71.3%, P< 0.05) and 50 microg/ml (79.3%,P< 0.02). Tenidap reduced IL-6 levels by 90.4% at 20 microg/ml (P< 0.005) and 96.0% (P< 0.05) at 50 microg/ml. Tenidap also reduced the synthesis of TNF-alpha by 66.9% (P< 0.05) and 77.4% at concentrations of 20 microg/ml and 50 microg/ml. Tenidap had a marked suppressive effect of over 90% (P< 0.0001) on PGE(2) synthesis in all three concentrations. Diclofenac (125 microg/l) decreased PGE(2) production by 95% (P< 0.0001), but had no significant effect in IL-1beta, IL-6, and TNF-alpha levels in the culture medium.Conclusion The ability to simultaneously suppress the release of proinflammatory cytokines and PGE(2) may help control osteolysis and prevent aseptic loosening of THR. This effect could increase implant longevity and lead the way to the pharmacological treatment of this pathology.     

Release of (1-->3)-beta-D-glucan from depth-type membrane filters and their in vitro effects on proinflammatory cytokine production

To clarify the origin of (1-->3)-beta-D-glucan in blood products and assess the biological activity of filter extracts, we evaluated (1-->3)-beta-D-glucan extraction from depth filters used to process blood products and their in vitro effects on proinflammatory cytokine production from macrophages. Cellulose or nylon filters were analyzed for (1-->3)-beta-D-glucan using the Fungitec G test. To evaluate the biological activity of the filter extracts, Mono Mac 6 cells (a human macrophage cell line) were cultured with filter extracts with or without lipopolysaccharide, and tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) concentrations in the culture media were measured. (1-->3)-beta-D-Glucan was released from seven cellulose filters but the nylon filter level was undetectable. Proinflammatory cytokine production ranged from 74.3% to 119.0% of the control for TNF-alpha and 81.2% to 115.9% for IL-1beta. TNF-alpha and IL-1beta levels were low without lipopolysaccharide. The data indicate that (1-->3)-beta-D-glucan in blood products is contaminated with the depth filters and that these filter extracts modulate proinflammatory cytokine production from macrophages.