The cytoplasmic domain of tissue factor in macrophages augments cutaneous delayed-type hypersensitivity.

In addition to its procoagulant role, tissue factor (TF) has important coagulation-independent roles, including in inflammation. The cytoplasmic domain of TF has been implicated in some of these coagulation-independent roles, particularly cell signaling. To assess the contribution of the cytoplasmic domain of TF to cell-mediated adaptive immunity, the development of cutaneous delayed-type hypersensitivity (DTH) was studied in mice lacking the cytoplasmic domain of TF (TF(deltaCT/deltaCT) mice). DTH responses in sensitized mice were significantly attenuated in TF(deltaCT/deltaCT) mice, and leukocyte-endothelial cell interactions, assessed by intravital microscopy, were impaired significantly. Studies in chimeric mice, created by bone marrow transplantation, showed that the absence of the cytoplasmic domain of TF in leukocytes rather than endothelial cells was responsible for reduced DTH and leukocyte recruitment. DTH responses to OVA could be induced in wild-type mice but not in TF(deltaCT/deltaCT) mice by transfer of activated CD4(+) OVA-specific TCR transgenic T cells, demonstrating that the defective DTH response in TF(deltaCT/deltaCT) mice was independent of any defect in T cell activation. Macrophage and neutrophil accumulation and expression of TNF-alpha mRNA and phospho-p38-MAPK were reduced significantly in TF(deltaCT/deltaCT) mice, and their macrophages had reduced P-selectin-binding capacity and reduced in vivo emigration in response to MCP-1. These results indicate that leukocyte expression of the cytoplasmic domain of TF contributes to antigen-specific cellular adaptive immune responses via effects on leukocyte recruitment and activation.

Urinary interleukin-18 does not predict acute kidney injury after adult cardiac surgery: a prospective observational cohort study.

INTRODUCTION: Urinary interleukin-18 (IL-18) measured during the immediate postoperative period could be a promising predictor of acute kidney injury following adult cardiac surgery. METHODS: In a single-centre prospective observational cohort study, we enrolled 100 adult cardiac surgical patients undergoing cardiopulmonary bypass at a tertiary hospital. We measured the urinary concentration of IL-18 and creatinine preoperatively, on arrival in the intensive care unit, and 24 hours postoperatively. We assessed urinary IL-18 concentration and urinary IL-18/urinary creatinine ratio in relation to the postoperative development of acute kidney injury defined as an increase in serum creatinine of greater than 50% from preoperative to postoperative peak value within 48 hours after surgery. RESULTS: Twenty patients developed acute kidney injury. On arrival in the intensive care unit and at 24 hours postoperatively, urinary IL-18 (median [interquartile range]) was not different in patients who subsequently developed acute kidney injury compared with those who did not: on arrival in the intensive care unit (168 [717] versus 104 [256] pg/mL; P = 0.70) and at 24 hours (195 [483] versus 165 [246] pg/mL; P = 0.47). On arrival in the intensive care unit (area under the curve for the receiver operating characteristic curve [AUC-ROCC] 0.53, 95% confidence interval [CI] 0.38 to 0.68; P = 0.70) and at 24 hours postoperatively (AUC-ROCC 0.55, 95% CI 0.40 to 0.71; P = 0.48), urinary IL-18 was not better than chance in predicting acute kidney injury. All findings were confirmed when urinary IL-18 was adjusted for urinary creatinine. Urinary IL-18 correlated with duration of cardiopulmonary bypass (P < 0.001). CONCLUSION: In adults, early postoperative measurement of urinary IL-18 appears not to be valuable in identifying patients who develop acute kidney injury after cardiac surgery, but rather represents a nonspecific marker of cardiopulmonary bypass-associated systemic inflammation.

Hemodialysis membrane with a high-molecular-weight cutoff and cytokine levels in sepsis complicated by acute renal failure: a phase 1 randomized trial.

BACKGROUND: Sepsis is the leading cause of acute renal failure. Intermittent hemodialysis (IHD) is a common treatment for patients with acute renal failure. However, standard hemodialysis membranes achieve only little diffusive removal of circulating cytokines. Modified membranes may enable both successful IHD treatment and simultaneous diffusive cytokine removal. STUDY DESIGN: Double-blind, crossover, randomized, controlled, phase 1 trial. SETTING & PARTICIPANTS: Tertiary intensive care unit. 10 septic patients with acute renal failure according to RIFLE class F. INTERVENTION: Each patient was treated with 4 hours of high-cutoff (HCO)-IHD and 4 hours of high-flux (HF)-IHD. OUTCOMES & MEASUREMENTS: We chose relative change in plasma interleukin 6 (IL-6) concentrations from baseline to 4 hours as the primary outcome for effective cytokine removal. We measured plasma and effluent concentrations of cytokines (IL-6, IL-8, IL-10, and IL-18) and albumin. RESULTS: Median age was 53 years (25(th) to 75(th) percentiles, 43 to 71 years). Both treatments achieved equal control of uremia. Four hours of HCO-IHD accomplished a greater decrease in plasma IL-6 levels (-30.3%) than 4 hours of HF-IHD (1.1%; P = 0.05). HCO-IHD, but not HF-IHD, achieved substantial diffusive clearance of several cytokines (IL-6, 14.1 mL/min; IL-8, 75.2 mL/min; and IL-10, 25.5 mL/min). Such clearance also was associated with greater relative decreases in plasma IL-8 and IL-10 levels in favor of HCO-IHD (P = 0.02, P = 0.04). We found significantly greater relative changes from prefilter to postfilter plasma IL-6, IL-8, and IL-10 values in favor of HCO-IHD (P = 0.02, P = 0.01, P < 0.01). During HCO-IHD, cumulative albumin loss into the effluent was 7.7 g (25(th) to 75(th) percentiles, 4.8 to 19.6) versus less than 1.0 g for HF-IHD (P < 0.01). LIMITATIONS: Small phase 1 trial. CONCLUSION: In septic patients with acute renal failure, HCO-IHD achieved simultaneous uremic control and diffusive cytokine clearances and a greater relative decrease in plasma cytokine concentrations than standard HF-IHD.

Cytokine dialysis: an ex vivo study.

To test the hypothesis that dialysis using a new large pore membrane would achieve effective cytokine removal, blood from six volunteers was incubated with endotoxin (1 mg) and then circulated through a closed circuit with a polyamide membrane (nominal cut-off: 100 kDa). Hemodialysis was conducted at 1 or 9 L/hr of dialysate flow at the start of circulation and after 2 and 4 hours. The peak dialysate/plasma concentration ratios were 0.92 for interleukin (IL)-1beta, 0.67 for IL-6, 0.94 for IL-8, 0.33 for tumor necrosis factor (TNF)-a, and 0.11 for albumin. The dialysate/plasma ratios for all cytokines and albumin were decreased with increased dialysate flow from 1 to 9 L/hr (p < 0.05). Clearances for IL-1beta, IL-6, and IL-8, however, were significantly improved with increased dialysate flow (p < 0.01). There was no increase in TNF-a clearance (not significant) and a decrease in albumin clearance (p < 0.01). The peak clearance at 9 L/hr was 33 ml/min for IL-1beta, 19 for IL-6, 51 for IL-8, 11 for TNF-alpha, and 1.2 for albumin. No adsorption of cytokines was observed. We conclude that cytokine dialysis is achievable through a membrane with a high cut-off point with negligible albumin loss. These findings support the technical feasibility of this new approach to blood purification in sepsis.

Protease-activated receptor-2 augments experimental crescentic glomerulonephritis.

Protease-activated receptor-2 (PAR-2) is a cellular receptor expressed prominently on epithelial, mesangial, and endothelial cells in the kidney and on macrophages. PAR-2 is activated by serine proteases such as trypsin, tryptase, and coagulation factors VIIa and Xa. It induces pleiotropic effects including vasodilatation, increasing plasminogen activator inhibitor (PAI-1) expression, mesangial cell proliferation, and cytokine production by macrophages. The role of PAR-2 in renal inflammation was studied in antiglomerular basement membrane antibody-induced crescentic glomerulonephritis (CGN) using PAR-2-deficient (PAR-2(-/-)) mice and wild-type littermate controls. PAR-2(-/-) mice had reduced crescent formation, proteinuria, and serum creatinine compared with wild-type mice 21 days after initiation of CGN. Glomerular accumulation of CD4(+) T cells and macrophages and the number of proliferating cells in glomeruli were similar in both groups. Glomerular fibrin deposition was significantly reduced in PAR-2(-/-) mice, and this was associated with reduced renal plasminogen activator inhibitor expression and increased renal matrix-metalloprotinase-9 activity. These results demonstrate a proinflammatory role for PAR-2 in CGN that is independent of effects on glomerular leukocyte recruitment and mesangial cell proliferation. PAR-2-mediated augmentation of renal plasminogen activator inhibitor expression and inhibition of matrix-metalloprotinase-9 activity may contribute to increased glomerular fibrin accumulation and glomerular injury in CGN.

The role of interleukin-4 and interleukin-12 in the progression of atherosclerosis in apolipoprotein E-deficient mice.

Accumulation of T cells and macrophages in atherosclerotic plaques and the formation of antibodies directed against plaque proteins suggests that adaptive immunity contributes to the development of atherosclerosis. The contribution of Th1 and Th2 helper cell subsets to atherogenesis was studied in a murine model by interbreeding apolipoprotein E-deficient (apoE(-/-)) mice with mice deficient in key cytokines that drive either Th1 responses [interleukin (IL)-12] or Th2 responses (IL-4). Compared to apoE(-/-) mice, apoE(-/-)/IL-12(-/-) mice had a 52% reduction in plaque area in the aortic root at 30 weeks of age (P < 0.001). ApoE(-/-)/IL-4(-/-) mice had a 27% reduction in plaque area compared to apoE(-/-) mice (P < 0.05) at 30 weeks of age, but their plaques were significantly larger than in apoE(-/-)/IL-12(-/-) mice at this stage (P < 0.05). By 45 weeks of age, there were no significant differences in lesion sizes in the aortic root between the strains, however apoE(-/-)/IL-4(-/-) mice showed a 58% and 64% decrease in disease in their aortic arch compared to apoE(-/-) (P < 0.05) and apoE(-/-)/IL-12(-/-) (P < 0.05) mice, respectively, and a 78% decrease in thoracic lesions compared to apoE(-/-)/IL-12(-/-) (P < 0.05). This suggests that both Th1 and Th2 cytokines play roles throughout the development of atherosclerosis in various vascular sites in apoE(-/-) mice.

The effect of coupled haemofiltration and adsorption on inflammatory cytokines in an ex vivo model.

BACKGROUND: The objective of this study was to evaluate the ex vivo removal of cytokines with an extracorporeal circuit using coupled large-pore haemofiltration and sorbent adsorption. METHODS: The setting for this study was a laboratory attached to the Intensive Care Unit of a tertiary hospital. Six healthy volunteers donated blood, which was incubated with endotoxin. Control blood was left at room temperature. Treatment blood was recirculated for 6 h through a closed circuit with a large-pore polysulfone haemofilter (average pore size 150 kDa) and an activated charcoal cartridge. Blood and ultrafiltrate were sampled hourly from three sites (pre-haemofilter for the circulating concentration, at cartridge inlet and cartridge outlet) to measure the concentrations of interleukins (IL)-1beta, -6, -8 and -10, and tumour necrosis factor (TNF). RESULTS: Control cytokine concentrations remained the same or increased slightly. Most of the preformed circuit cytokines were removed, with the exception of IL-10. The average sieving coefficients were 0.61 for IL-1beta, 1.34 for IL-6, 0.30 for IL-8, and 0.56 for TNF. Average single-pass clearances were 49, 107, 24 and 45 ml/min, respectively. The cartridge adsorbed 90% of IL-1beta, 72% of IL-6, 100% of IL-8, and 7% of TNF during each pass. CONCLUSION: The combination of a large-pore haemofilter and charcoal cartridge removed several cytokines efficiently under ex vivo conditions. This technique can now be tested for cytokine removal in vivo.

A phase II randomized, controlled trial of continuous hemofiltration in sepsis.

OBJECTIVE: To study the effect of early and continuous venovenous hemofiltration (CVVH) on the plasma concentrations of several humoral mediators of inflammation and subsequent organ dysfunction in septic patients. DESIGN: Randomized, controlled trial. SETTING: Intensive care unit of a tertiary hospital. PATIENTS: Twenty-four patients with early septic shock or septic organ dysfunction. INTERVENTIONS: Random allocation to receive 48 hrs of isovolemic CVVH at 2 L/hr of fluid exchange or no hemofiltration. MEASUREMENTS AND MAIN RESULTS: We measured the plasma concentrations of complement fractions C3a and C5a, interleukins 6, 8, and 10, and tumor necrosis factor alpha at baseline and 2, 24, 26, 48, and 72 hrs. A multiple organ dysfunction score (MODS) was calculated daily for each patient until death or discharge from the intensive care unit. The concentrations of most mediators decreased between baseline and 72 hrs. Some significant falls in concentration could be identified between specific time points, but CVVH was not associated with an overall reduction in any plasma cytokine concentrations. There was also no difference between the mean cumulative MODS for control survivors (43.3 +/- 19.7) and CVVH survivors (33.2 +/- 19.0; p = .30), and no difference between the average MODS calculated for all controls (4.1 +/- 1.9) and all CVVH subjects (3.3 +/- 1.7; p = .26). CVVH did not improve oxygenation, lower the platelet count, or reduce the duration of vasopressor support and mechanical ventilation. CONCLUSIONS: Early use of CVVH at 2 L/hr did not reduce the circulating concentrations of several cytokines and anaphylatoxins associated with septic shock, or the organ dysfunction that followed severe sepsis. CVVH using current technology cannot be recommended as an adjunct to the treatment of septic shock unless severe acute renal failure is present.

The cytoplasmic domain of tissue factor contributes to leukocyte recruitment and death in endotoxemia.

Tissue factor (TF) is an integral membrane protein that binds factor VIIa and initiates coagulation. The extracellular domain of TF is responsible for its hemostatic function and by implication in the dysregulation of coagulation, which contributes to death in endotoxemia. The role of the cytoplasmic domain of tissue factor in endotoxemia was studied in mice, which lack the cytoplasmic domain of TF (TF(deltaCT/deltaCT)). These mice develop normally and have normal coagulant function. Following i.p injection with 0.5 mg of lipopolysaccharide (LPS), TF(deltaCT/deltaCT) mice showed significantly greater survival at 24 hours compared to the wt mice (TF(+/+)). The serum levels of TNF-alpha and IL-1beta were significantly lower at 1 hour after LPS injection and IL-6 levels were significantly lower at 24 hours in TF(deltaCT/deltaCT) mice compared to TF(+/+)mice. Neutrophil recruitment into the lung was also significantly reduced in TF(deltaCT/deltaCT) mice. Nuclear extracts from tissues of endotoxemic TF(deltaCT/deltaCT) mice also showed reduced NFkappaB activation. LPS induced leukocyte rolling, adhesion, and transmigration in post-capillary venules assessed by intravital microscopy was also significantly reduced in TF(deltaCT/deltaCT) mice. These results indicate that deletion of the cytoplasmic domain of TF impairs the recruitment and activation of leukocytes and increases survival following endotoxin challenge.

Plasminogen activator inhibitor-1 is a significant determinant of renal injury in experimental crescentic glomerulonephritis.

Crescentic glomerulonephritis is characterized by glomerular fibrin deposition, and experimental crescentic glomerulonephritis has been shown to be fibrin-dependent. Net fibrin deposition is a balance between activation of the coagulation system causing glomerular fibrin deposition and fibrin removal by the plasminogen-plasmin (fibrinolytic) system. Plasminogen activator inhibitor-1 (PAI-1) inhibits fibrinolysis by inhibiting plasminogen activators and has effects on leukocyte recruitment and matrix deposition. To test the hypothesis that the presence of PAI-1 and its levels were a determinant of injury in crescentic glomerulonephritis, accelerated anti-glomerular basement membrane glomerulonephritis was induced in mice genetically deficient in PAI-1 (PAI-1 -/-), PAI-1 heterozygotes (PAI-1 +/-), and mice engineered to overexpress PAI-1 (PAI-1 tg). Compared with strain-matched genetically normal animals, PAI-1 -/- mice with glomerulonephritis developed fewer glomerular crescents, less glomerular fibrin deposition, fewer infiltrating leukocytes, and less renal collagen accumulation at day 14 of disease. The reduction in disease persisted at day 28, when injury had become more established. In contrast, mice overexpressing the PAI-1 gene (PAI-1 tg), that have basal plasma and renal PAI-1 levels several times, normal developed increased glomerular crescent formation, more glomerular fibrin deposition, increased numbers of infiltrating leukocytes, and more renal collagen at both time points. These studies demonstrate that PAI-1 is a determinant of glomerular fibrin deposition and renal injury in crescentic glomerulonephritis.