Transforming growth factor-beta is involved in the pathogenesis of dialysis-related amyloidosis

BACKGROUND: Advanced glycation end product-modified beta2-microglobulin (AGE-beta2m) is an important component of dialysis-related amyloidosis (DRA). Its presence induces monocyte chemotaxis and the release of the proinflammatory cytokines through macrophage activation. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that also has chemotactic activity for monocytes at very low (0.1 to 10 pg/mL) concentrations and inhibits proinflammatory cytokine production of macrophages. In this study, we investigated the role of TGF-beta in the pathogenesis of DRA. METHODS: We performed an immunohistochemical study of DRA tissues (8 cases) to confirm the existence of TGF-betas and their receptors; we also performed a chemotaxis assay of human monocytes as well as enzyme-linked immunosorbent assay (ELISA) of TGF-beta1, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-1 receptor antagonist (IL-1Ra) in the supernatant of human monocyte-derived macrophage cell culture under varying conditions of incubation with TGF-beta1, AGE-beta2m, and TGF-beta1 antibody additions. RESULTS: There was positive staining for TGF-betas (types 1, 2, and 3) and their receptors (types I, II, and III) in infiltrated macrophages (CD68+), synovial lining cell, as well as vascular walls around amyloid deposition. AGE-beta2m also induced TGF-beta1 production by macrophages in a dose-dependent manner (410 +/- 80 pg/mL at 12.5 microg/mL, 621 +/- 62 pg/mL at 25 microg/mL, and 776 +/- 62 pg/mL at 50 microg/mL of AGE-beta2m). AGE-beta2m induced significant TNF-alpha and IL-1Ra production by macrophage. The addition of exogenous TGF-beta1 (0.1 to 10 ng/mL) decreased AGE-beta2m-induced TNF-alpha production and increased IL-1Ra production in a dose-dependent fashion. IL-1beta production was not effected by any experimental conditions. In chemotaxis assay, anti-TGF-beta1 antibody (0.1 to 10 microg/mL) attenuated AGE-beta2m-induced monocyte chemotaxis. CONCLUSIONS: These results provide the first evidence to our knowledge for the presence of TGF-beta in DRA tissue, as well as the stimulatory action of AGE-beta2m on tissue macrophages. In turn, TGF-beta suppresses the proinflammatory activation of macrophages, suggesting a dual role for TGF-beta in the inflammatory process of DRA. These observations may provide a pathophysiologic link between TGF-beta and DRA.     

Impaired mitogen-activated protein kinase activation and altered cytokine secretion in endotoxin-tolerant human monocytes

BACKGROUND: Dysregulation of monocyte/macrophage cytokine production after exposure to multiple inflammatory stimuli may contribute to multiple organ failure and sepsis. Endotoxin (lipopolysaccharide [LPS]) activation of murine macrophage results in the phosphorylation of kinases in the mitogen-activated protein kinase cascade. Pretreatment of murine macrophages with LPS induces LPS-tolerance, with inhibition of LPS-stimulated activation of kinases (ERK1/2 and p38) and diminished release of tumor necrosis factor (TNF). We sought to determine whether similar alterations in LPS-dependent signal transduction are present in LPS-tolerant human peripheral blood monocytes. METHODS: Human peripheral blood monocytes from healthy volunteer donors (n = 12) were incubated in RPMI 1640 culture medium +/- 10 ng/mL of LPS for 18 hours, then stimulated with 0 to 1,000 ng/mL of LPS. Supernatant TNF and interleukin-1 (IL-1) levels were measured after 5 hours by enzyme-linked immunosorbent assay. Activation of the p42/p44 kinases (ERK1/2) was measured 15 minutes after LPS with monoclonal antibodies to diphosphorylated (active) ERK1/2 using novel flow cytometric methods. RESULTS: LPS-tolerant (10 ng/mL LPS pretreatment) human monocytes had significant inhibition of LPS-stimulated TNF secretion but augmented IL-1 release (p < 0.05). Nontolerant human monocytes had a dramatic increase in the percentage of ERK1/2-positive cells in response to an initial stimulation with LPS. This did not occur in the LPS-tolerant cells. Phorbol-12-myristate-13 acetate restored ERK1/2 activation in LPS-tolerant human monocytes. CONCLUSION: LPS-tolerance in human monocytes is associated with inhibition of LPS-stimulated TNF secretion, augmented release of IL-1, and defective activation of mitogen-activated protein kinase cascade (ERK1/2). These results suggest a method of identifying LPS-tolerance and monocyte dysfunction in patients with sepsis.     

Interleukin-1 beta and tumor necrosis factor-alpha release in normal and diseased human infrarenal aortas.

The presence of chronic inflammatory cells in the adventitia and media of abdominal aortic aneurysms and aortic occlusive disease suggest an immunologic response. The purpose of this study is to determine whether normal or diseased infrarenal aortas liberate the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). Twenty-six infrarenal aortic biopsies (5 aortic occlusive disease, 15 abdominal aortic aneurysms, and 6 cadaveric donors) were weighed, minced into small pieces, and incubated in media for 48 hours. Conditioned media was harvested at 48 hours and assayed for IL-1 beta or TNF-alpha with use of an ELISA assay. Comparison of groups was performed with a one-way analysis of variance. The constitutive IL-1 beta produced by abdominal aortic aneurysms was significantly different than that in cadaveric donors (908 +/- 194 pg/ml [SE] vs 100 +2- 30 pg/ml). There was no statistically significant difference between abdominal aortic aneurysms and aortic occlusive disease (908 +/- 194 pg/ml vs 604 +/- 256 pg/ml) or aortic occlusive disease and cadaveric donor (604 +/- 256 vs 100 +/- 30). In time-course studies for the release of IL-1 beta, abdominal aortic aneurysms demonstrated maximal release at 48 hours. IL-1 beta release was augmented by lipopolysaccharide in all categories. A dose response curve demonstrated maximal IL-1 beta release on stimulation with 5 micrograms/ml LPS. Constitutive TNF-alpha production was low, ranging from 13 +/- 1.5 pg/ml in cadaveric donor, to 20 pg/ml in aortic occlusive disease, and 24 +/- 11 pg/ml in abdominal aortic aneurysms. There was no augmentation in TNF-alpha with lipopolysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for interleukin-1 beta production by cultured normal human osteoblast-like cells.

To determine if bone cells produce interleukin-1 beta (IL-1 beta), a potent bone resorption-stimulating agent, we studied well-characterized, nearly homogeneous cultures of normal human osteoblast-like (hOB) cells. With four strains of such cells, vehicle-treated cultures produced minimal IL-1 beta (mean +/- SEM, 1.3 +/- 0.3 pg/ml per 10(6) cells per 24 h) and showed dose-dependent (r = 0.99) increases to 2.2 +/- 0.7, 5.0 +/- 0.9, or 17.8 +/- 6.7 pg/ml, respectively, after treatment with lipopolysaccharide (LPS) at 3, 10, or 30 micrograms/ml (for increases after 10 and 30 micrograms/ml treatments, P less than 0.05). After treatment with tumor necrosis factor alpha (TNF-alpha) at 10 U/ml, IL-1 beta increased to 16.2 +/- 3.7 pg/ml (P less than 0.05). Neither 17 beta-estradiol nor bovine parathyroid hormone(1-34) (each at 10 nM), alone or in combination with LPS or TNF-alpha, affected IL-1 beta release. Northern blot analysis of total cellular RNA preparation revealed a single hybridization band at 1.9 kb when probed with a partially deleted cDNA for human IL-1 beta. The steady-state IL-1 beta mRNA levels showed a significant increase with LPS treatment and a lesser increase with TNF-alpha treatment in hOB cells. Moreover, TNF-alpha produced an even greater increase in IL-1 mRNA in HOBIT cells, a well-differentiated clonal cell line derived from normal hOB cells transfected with the SV40 large T antigen. We conclude that human cells of the osteoblast lineage produce IL-1 beta in response to well-recognized stimuli for IL-1 release from responsive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Irradiation with 780 nm diode laser attenuates inflammatory cytokines but upregulates nitric oxide in lipopolysaccharide-stimulated macrophages: implications for the prevention of aneurysm progression

BACKGROUND AND OBJECTIVES: Low level laser irradiation (LLLI) has been shown to reduce inflammation in a variety of clinical situations. We have shown that LLLI (780 nm) increases aortic smooth muscle cell proliferation and matrix protein secretion and modulates activity and expression of matrix metalloproteinases. Inflammation is a major component of arteriosclerotic diseases including aneurysm. Macrophage recruitment and secretion of pro-inflammatory cytokines and the vasodilator, nitric oxide (NO), are central to most immune responses in the arterial wall. The present study was designed to determine the effect of LLLI on cytokine gene expression and secretion as well as gene expression of inducible nitric oxide synthase (iNOS) and NO production in lipopolysaccharide (LPS)-stimulated macrophages. STUDY DESIGN/MATERIALS AND METHODS: Murine monocyte/macrophages (RAW 264.7) were irradiated with a 780 nm diode laser (2 mW/cm(2), 2.2 J/cm(2)) during stimulation with LPS (0, 0.1, and 1 microg/ml). Gene expression of chemokines, cytokines, and iNOS were assessed by RT-PCR. Secretion of interleukin (IL)-1beta and monocyte chemotactic protein (MCP)-1 and NO were assessed by ELISA and the Griess reaction, respectively. RESULTS: LLLI reduced gene expression of MCP-1, IL-1alpha, IL-10 (P<0.01), IL-1beta, and IL-6 (P<0.05) when cells were stimulated by 1 microg/ml LPS. LLLI reduced LPS-induced secretion of MCP-1 over non-irradiated cells by 17+/-5% and 13+/-5% at 12 hours (0.1 and 1 microg/ml LPS; P<0.01 and P=0.05, respectively), and reduced IL-1beta by 22+/-5% and 25+/-9% at 24 hours (0.1 and 1 microg/ml LPS, P=0.01 and P=0.06, respectively). However, LLLI increased NO secretion after 12 hours (LLLI vs. Control: without LPS, 1.72+/-0.37 vs. 0.95+/-0.4 microM, P<0.05; 0.1 microg/ml LPS, 7.46+/-1.62 vs. 4.44+/-1.73 microM, P=0.06; 1 microg/ml LPS, 10.91+/-3.53 vs. 6.88+/-1.52 microM, P<0.05). CONCLUSIONS: These properties of LLLI, with its effects on smooth muscle cells reported previously, may be of profound therapeutic relevance for arterial diseases such as aneurysm where inflammatory processes and weakening of the matrix structure of the arterial wall are major pathologic components.     

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.     

Effect of a novel adsorbent on cytokine responsiveness to uremic plasma

BACKGROUND: Middle molecules such as beta2-microglobulin (beta2M) and advanced glycation end products (AGE)-modified proteins contribute to inflammation in uremia. The BetaSorb column is a new adsorptive device, which contains copolymeric beads, suitable for removal of beta2M and other middle molecules. We assessed the effect of this column on the bioreactivity of uremic plasma, as measured by cytokine responsiveness. METHODS: Uremic plasma was perfused in vitro through the column (10 mL/min) and samples were collected after 10 to 30 passes. Endotoxin-stimulated tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production by THP-1-derived monocytes was measured following brief exposure to uremic plasma. beta2M levels were measured. The contribution of AGE-modified proteins to the bioreactivity of uremic plasma was explored. RESULTS: TNF-alpha and IL-10 production markedly decreased after 30 passes (629 +/- 78 vs. 144 +/- 62 pg/mL; 207 +/- 25 vs. 117 +/- 23 pg/mL; P=0.04). The column removed beta2M efficiently with a marked decline in plasma levels by 99% after 30 passes. Neutralization of AGE receptor (RAGE) resulted in a further reduction in the bioreactivity of uremic plasma. This was observed with nonperfused, as well as perfused, uremic plasma, suggesting that AGE-modified proteins were biologically active and still present after perfusion. CONCLUSION: The sorbent beads removed uremic solute(s) that prime monocytes to enhanced cytokine production. Removal of beta2M was efficient, and of native and AGE-modified middle molecules likely.     

Isoproterenol inhibits bacterial lipopolysaccharide-stimulated release of tumor necrosis factor-alpha from human heart tissue

Recent evidence suggests that inflammatory cytokines, particularly tumor necrosis factor alpha (TNF-alpha), may play a role in heart disease. Elevated plasma levels of the cytokine have been reported in congestive heart failure and severe angina and after myocardial infarction. The exact role of TNF-alpha in heart disease and how production is stimulated and regulated in the heart are current areas of investigation. Regarding regulation of production, isoproterenol elevates cyclic AMP and inhibits TNF-alpha release in macrophages. Therefore we hypothesized that stimulation of beta-adrenergic receptors of the sympathetic nervous system would inhibit release of the cytokine from heart tissue. With Institutional Review Board approval and patient consent atrial tissue was obtained during preparation for cardiac bypass. The tissue was divided into segments, placed in culture medium, and incubated for various times in the presence or absence of lipopolysaccharide (LPS) (20 microg/mL) and/or isoproterenol (1 microM). The medium was removed and analyzed for biologically active TNF-alpha by the L929 cell cytotoxicity assay. Tissue samples were weighed and TNF-alpha release was expressed as pg TNF-alpha/mg tissue. Initially, to determine the time course of release, measurements were made at 2, 5, 10, 15, 30, 60, 120, 180, and 360 minutes after the addition of LPS. Elevated TNF-alpha levels in the culture medium were reliably detected at 360 minutes after exposure to LPS. In atrial tissue obtained from seven patients TNF-alpha released into the culture medium at 360 minutes was 6 +/- 3 pg/mg tissue. In the presence of LPS, levels of the cytokine in the culture medium increased to 604 +/- 233 pg/mg tissue (P < 0.05 vs LPS alone). When isoproterenol and LPS were simultaneously added to the culture medium release of TNF-alpha was reduced by 87 per cent to 82 +/- 40 pg/mg tissue (P < 0.05 vs LPS alone). Our results show that activation of the beta-adrenergic receptor inhibits myocardial production of TNF-alpha. This finding suggests that the sympathetic nervous system inhibits production of the cytokine and that impaired sympathetic function in heart failure may play a role in the elevated levels of TNF-alpha.     

Interleukin-1beta induces human proximal tubule cell injury,alpha-smooth muscle actin expression and fibronectin production

BACKGROUND: Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS: Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS: IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION: IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.     

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.     

Urinary and renal interstitial concentrations of TNF-alpha increase prior to the rise in albuminuria in diabetic rats

BACKGROUND: The development of diabetic nephropathy has been linked to the release of vasoactive hormones and growth factors. Currently the role of inflammatory cytokines in this pathogenic process is not clear. METHODS: We utilized the microdialysis technique to monitor early changes in tumor necrosis-alpha (TNF-alpha) levels in the renal interstitial fluid and urine of conscious Sprague-Dawley rats (N = 8) before and after induction of diabetes with streptozotocin (STZ). Measurement of the urinary albumin excretion (UAE) was utilized to monitor the development and progression of diabetic nephropathy. RESULTS: UAE increased from 0.56 +/- 0.20 microg/min to 8.14 +/- 2.98 microg/min 17 days after induction of diabetes (P = 0.01). Renal interstitial fluid TNF-alpha increased from 11.96 +/- 5.32 pg/mL at baseline to 45.02 +/- 11.69 pg/mL 5 days after induction of diabetes (P = 0.03). Renal interstitial fluid TNF-alpha levels remained elevated throughout the remainder of the study period. Urinary TNF-alpha also increased significantly compared to baseline 3 days after induction of diabetes (294.18 +/- 36.94 pg/mL vs. 16.05 +/- 6.07 pg/mL, P < 0.002). There was a second significant rise in urinary TNF-alpha concentration to 638.16 +/- 36.94 pg/mL 21 days after induction of diabetes (P < 0.001). Serum TNF-alpha levels were undetectable before STZ injection and remained undetectable by the end of the study. Urinary and renal interstitial fluid TNF-alpha in the control rats (N = 5) did not change throughout the study. CONCLUSION: We found an early rise in renal TNF-alpha levels after induction of diabetes with STZ, which precedes the rise in UAE by about 2 weeks. These findings suggest a possible contribution of TNF-alpha in the complicated pathogenic process resulting in microalbuminuria in diabetes.     

Effect of hemoperfusion with polymyxin B-immobilized fiber on plasma endothelin-1 and endothelin-1 mRNA in monocytes from patients with sepsis

Hemoperfusion using polymyxin B-immobilized fiber (PMX-F) is reported to be an effective treatment for sepsis. The aim of the present study is to assess whether plasma endothelin-1 (ET-1) and ET-1 messenger RNA (mRNA) levels in peripheral-blood monocytes are altered in patients with sepsis and whether PMX-F treatment affects plasma ET-1 and monocyte ET-1 mRNA levels. Sixteen patients with sepsis and 20 healthy volunteers were included in this study. Plasma ET-1 concentration was measured by radioimmunoassay (RIA), and plasma levels of transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta) were measured by enzyme-linked immunosorbent assay (ELISA). Sixteen patients with sepsis were treated with direct hemoperfusion using PMX-F columns. Blood endotoxin levels decreased significantly from 35 to 10 pg/mL after two treatments of direct hemoperfusion, each for 2 hours. Patients with sepsis showed significantly increased levels of plasma ET-1 (P < 0.001) and monocyte ET-1 mRNA (P < 0.001) compared with healthy volunteers. However, no differences in plasma levels of TGF-beta, TNF-alpha, and IL-1beta existed between patients with sepsis and healthy volunteers. Increased plasma ET-1 levels and monocyte ET-1 mRNA levels in patients with sepsis decreased significantly after PMX-F treatment (P < 0.01). These data suggest that the secretion of ET-1 from peripheral-blood monocytes may be stimulated by endotoxin, and PMX-F treatment may be effective in reducing ET-1 secretion in patients with sepsis.     

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.     

In vitro modulation of monocyte chemoattractant protein-1 release in human pancreatic islets

Islet transplantation is a new approach to treat type 1 diabetic patients. Despite its great potential and progressively increasing success rate, islet engraftment still represents an unsolved problem. Only part of the transplanted beta-cell mass survives after infusion due to hypoxia and inflammatory reactions, principally mediated by macrophages. We have demonstrated that human islets release monocyte chemoattractant protein-1 (MCP-1), one of the most powerful macrophage chemokines, which may impair the fate of a transplant. In this study we have attempted to modulate in vitro MCP-1 release by human islets. Human islets isolated using the automated method were cultured in CMRL or M199 standard culture media alone or supplemented with (1) two intracellular kinase inhibitors (10 micromol/L RO8220, a protein kinase C inhibitor, and rcAMP 20 micromol/L, a protein kinase A inhibitor) or (2) two antioxidant and cell-protective agents (vitamin E, vitamin B); or (3) immunosuppressive drugs (0.001 to 10 ng/mL cyclosporine, 0.1 to 100 ng/mL rapamycin, 0.1 to 10 ng/mL tacrolimus, 0.001 to 10 ng/mL mycophenolate acid). We observed that the only culture condition that significantly decreased MCP-1 in human islets were CMRL (31 +/- 12 in CMRL vs 539 +/- 184 pg/mL, in M199, P <.05) or cyclosporine (514 +/- 83 pg/mL in control islet vs 307 +/- 13, 231 +/- 44, 192 +/- 4, 242 +/- 113, 169 +/- 15 pg/mL in islet plus cyclosporine ranging from 0.001 to 10 ng/mL, respectively, P >.05). The capacity of in vitro factors to decrease human islet MCP-1 release suggests strategies to increase the success of islet transplantation.     

Effects of activated protein C on the mesenteric microcirculation and cytokine release during experimental endotoxemia

PURPOSE: Activated protein C (APC) is the first anti-inflammatory drug to be approved for the treatment of severe sepsis. However, the underlying mechanisms are not completely elucidated. Therefore, the aim of our study was to evaluate the effects of APC on the microcirculation (mesenteric leukocyte-endothelial interaction, plasma extravasation) using intravital microscopy (IVM) and on cytokine release during experimental endotoxemia in rats. METHODS: We divided forty, male, Lewis rats into four groups (n = 10 per group): Controls, LPS (15 mg x kg(-1) lipopolysaccharide iv), APC (2 mg x kg(-1) APC iv), and LPS+APC. We determined mesenteric leukocyte-endothelial interactions and plasma extravasation at zero, one and two hours following administration of LPS and APC by IVM. Plasma levels of tumour necrosis factor-alpha, IL-1beta, interleukin (IL)-6, and IL-10 were measured at zero and at two hours. RESULTS: Leukocyte adherence (-74%) and plasma extravasation (-28%) during endotoxemia were diminished significantly following APC treatment, compared to untreated LPS animals (P = 0.0001 and P = 0.0004, respectively). Interleukin-1ss release was also significantly reduced by APC treatment (2567.4 +/- 320.9 pg x mL(-1) in the LPS group vs 1626.1 +/- 427.2 pg x mL(-1) in the LPS+APC group; P = 0.001).Conclusion: These rodent experiments showed that APC treatment significantly attenuated deterioration of the mesenteric microcirculation and systemic IL-1ss release caused by endotoxin challenge. Because of the crucial role of the microcirculation in ongoing sepsis pathogenesis and multiple organ dysfunction syndrome, these effects may be of clinical importance.     

The effect of 17beta-estradiol on production of cytokines in cultures of peripheral blood

Estrogen's action on bone may be mediated by cytokines produced by monocytes. We have reported a decreased ratio of interleukin-1beta (IL-1beta) to interleukin-1 receptor antagonist (IL-1ra) produced by whole blood cultures in vivo in women taking hormone replacement therapy (HRT). Also, one study has shown an effect of estradiol on tumor necrosis factor-alpha (TNF-alpha) secretion by separated monocytes in vitro. The aim of this study was to evaluate the effect of estrogen in vitro on the secretion of cytokines using whole blood cultures. Subjects consisted of 12 healthy postmenopausal women, ages 57-69 years, 4-20 years since menopause. Cytokines IL-1beta, interleukin-1alpha (IL-1alpha), IL-1ra, interleukin-6 (IL-6), TNF-alpha, and granulocyte macrophage-colony stimulating factor (GM-CSF) were measured in unstimulated and in stimulated (500 ng/mL lipopolysaccharide [LPS]) whole blood cultures treated with 17beta-estradiol (E(2)) at concentrations of 10(-12)--10(-6) mol/L. We found significant decreases in the spontaneous secretion of IL-6, TNF-alpha, IL-1ra, IL-1beta, and ratio of IL-1beta/IL-1ra compared with control, at physiological concentrations of E(2). The action of E(2) was blocked by the use of the antiestrogen ICI 182780 in coculture. A decrease in cytokine secretion was not observed when the inactive form of estrogen, 17alpha-estradiol, was used in place of 17beta-estradiol. GM-CSF and IL-1alpha were not detectable in unstimulated cultures. Cytokine levels measured in stimulated cultures were not attenuated by treatment with E(2). We conclude that E(2) inhibits the spontaneous secretion of cytokines measured in whole blood cultures at physiological concentrations, and that the powerful stimulatory effect of LPS prevents any significant inhibition by E(2) in stimulated cultures.     

p38丝裂原活化蛋白激酶信号转导通路和核因子-κB/抑制因子κB通路在烧伤后促炎性细胞因子产生中的相互作用

目的探讨烧伤后p38丝裂原活化蛋白激酶(MAPK)信号转导通路和核因子(NF)-κB/抑制因子(I)κB通路在调控细胞因子产生方面的地位及是否存在相互作用。方法人单核细胞株THP-1分为正常血清对照组、烧伤血清组、烧伤血清+SB203580组(p38 MAPK特异性抑制剂)和烧伤血清+PDTC(NF-κB特异性抑制剂)组,每组均实验8次。血清刺激24 h后,酶联免疫吸附法测定上清液中肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的含量,蛋白印迹(W estern b lot)法检测THP-1内p38 MAPK的活性和IκBα的表达,凝胶电泳迁移率变化分析法检测THP-1内NF-κB和激活蛋白(AP-1)活性的变化。结果和正常血清相比,烧伤血清刺激后24 h,THP-1上清液中TNF-α和IL-1β的含量均明显上升[分别为(7.30±0.84)ng/m l和(2.20±0.28)ng/m l,P<0.05;(2.88±0.38)ng/m l和(0.81±0.14)ng/m l,P<0.05],p38 MAPK活性升高(4728±582和1291±163,P<0.05),IκBα含量下降(1211±115和2658±318,P<0.05),THP-1中NF-κB活性和AP-1活性均较正常血清显著升高(1636±170和317±32,P<0.05;946±137和361±40,P<0.05),使用SB203580和PDTC均能抑制烧伤血清刺激后THP-1上清液中TNF-α和IL-1β的含量的上升。预先给予SB203580能抑制烧伤血清刺激后THP-1中p38 MAPK和AP-1活性升高,但对IκBα含量和NF-κB活性无显著影响;预先给予PDTC可以防止烧伤血清刺激后THP-1中IκBα含量的下降和NF-κB活性的升高,而对p38 MAPK和AP-1活性无影响。结论在烧伤后全身炎症反应的发生中,p38 MAPK信号转导通路和NF-κB/IκB通路是两个平行和独立的信号转导通路,彼此之间没有直接的联系,共同调节着烧伤后单核细胞TNF-α和IL-1β的产生。     

Intracellular monocyte cytokine production and CD 14 expression are up-regulated in severe vs mild chronic heart failure.

BACKGROUND: The role of circulating monocytes in the process of low-grade inflammation, characteristic of chronic heart failure (CHF), has recently been questioned. Lipopolysaccharide (LPS) desensitization has been proposed to mediate reduced monocyte cytokine elaboration in patients with severe CHF. METHODS: Intracellular monocyte production of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor (TNF)-alpha, and monocyte CD 14 expression were measured flow-cytometrically without and after 8-hour LPS stimulation in 46 patients with CHF and in a healthy control group. RESULTS: Basal cytokine concentrations were similar for the control and the mild CHF groups (New York Heart Association [NYHA] Class I or II). After LPS stimulation, IL-6 (p=0.002) and TNF-alpha levels (p=0.001) were lower in the latter group, whereas IL-1 beta production was comparable. For the moderate-severe CHF patients, unstimulated IL-1 beta (p=0.04) was higher, whereas IL-6 (p=0.2) and TNF-alpha (p=0.1) levels were not different from the controls. Measurement of LPS-stimulated cytokine production showed no differences between the control group and patients with moderate-severe CHF (all p= 0.5). Upon comparing mild vs moderate-severe CHF patients, higher levels of unstimulated cytokine production (IL-1 beta, p=0.002; IL-6, p=0.01; TNF-alpha, p=0.003), stimulated IL-1 beta (p=0.002) and IL-6 (p=0.008) were found in the latter patients. CD 14 expression in the moderate-severe CHF group was higher than in the mild-CHF group (p = 0.03) and was strongly related to stimulated IL-1 beta (r=0.62, p<0.0001), IL-6 (r=0.56, p=0.0002) and TNF-alpha (r=0.41, p=0.006) production. CONCLUSIONS: CD 14 expression and monocyte cytokine production, both unstimulated and after LPS stimulation, are increased in moderate-severe CHF when compared with mild CHF. These data suggest that circulating monocytes, possibly via increased CD 14 expression, may play a significant role in the immunologic dysbalance observed in advanced CHF.