The effect of aprotinin on hypoxia-reoxygenation-induced changes in neutrophil and endothelial function

BACKGROUND AND OBJECTIVE: An acute inflammatory response associated with cerebral ischaemia-reperfusion contributes to the development of brain injury. Aprotinin has potential, though unexplained, neuroprotective effects in patients undergoing cardiac surgery. METHODS: Human neutrophil CD11 b/CD18, endothelial cell intercellular adhesion molecule-1 (ICAM-1) expression and endothelial interleukin (IL)-1beta supernatant concentrations in response to in vitro hypoxia-reoxygenation was studied in the presence or absence of aprotinin (1600 KIU mL(-1)). Adhesion molecule expression was quantified using flow cytometry and IL-1beta concentrations by enzyme-linked immunosorbent assay. Data were analysed using ANOVA and post hoc Student-Newman-Keuls test as appropriate. RESULTS: Exposure to 60-min hypoxia increased neutrophil CD11b expression compared to normoxia (170+/-46% vs. 91+/-27%, P = 0.001) (percent intensity of fluorescence compared to time 0) (n = 8). Hypoxia (60 min) produced greater upregulation of CD11b expression in controls compared to aprotinin-treated neutrophils [(170+/-46% vs. 129+/-40%) (P = 0.04)] (n = 8). Hypoxia-reoxygenation increased endothelial cell ICAM-1 expression (155+/-3.7 vs. 43+/-21 mean channel fluorescence, P = 0.0003) and IL-1beta supernatant concentrations compared to normoxia (3.4+/-0.4 vs. 2.6+/-0.2, P = 0.02) (n = 3). Hypoxia-reoxygenation produced greater upregulation of ICAM- 1 expression [(155+/-3.3 vs. 116+/-0.7) (P = 0.001)] and IL-1beta supernatant concentrations [(3.4+/-0.3 vs. 2.6+/-0.1) (P = 0.01)] in controls compared to aprotinin-treated endothelial cell preparation (n = 3). CONCLUSIONS: Hypoxia-reoxygenation-induced upregulation of neutrophil CD11b, endothelial cell ICAM-1 expression and IL-1beta concentrations is decreased by aprotinin at clinically relevant concentrations.     

Activated endothelial interleukin-1beta, -6, and -8 concentrations and intercellular adhesion molecule-1 expression are attenuated by lidocaine

Endothelial cells play a key role in ischemia reperfusion injury. We investigated the effects of lidocaine on activated human umbilical vein endothelial cell (HUVEC) interleukin (IL)-1beta, IL-6, and IL-8 concentrations and intercellular adhesion molecule-1 (ICAM-1) expression. HUVECs were pretreated with different concentrations of lidocaine (0 to 0.5 mg/mL) for 60 min, thereafter tumor necrosis factor-alpha was added at a concentration of 2.5 ng/mL and the cells incubated for 4 h. Supernatants were harvested, and cytokine concentrations were analyzed by enzyme-linked immunosorbent assay. Endothelial ICAM-1 expression was analyzed by using flow cytometry. Differences were assessed using analysis of variance and post hoc unpaired Student's t-test where appropriate. Lidocaine (0.5 mg/mL) decreased IL-1beta (1.89 +/- 0.11 versus 4.16 +/- 1.27 pg/mL; P = 0.009), IL-6 (65.5 +/- 5.14 versus 162 +/- 11.5 pg/mL; P < 0.001), and IL-8 (3869 +/- 785 versus 14,961 +/- 406 pg/mL; P < 0.001) concentrations compared with the control. IL-1beta, IL-6, and IL-8 concentrations in HUVECs treated with clinically relevant plasma concentrations of lidocaine (0.005 mg/mL) were similar to control. ICAM-1 expression on lidocaine-treated (0.05 mg/mL) HUVECs was less than on controls (198 +/- 52.7 versus 298 +/- 50.3; Mean Channel Fluorescence; P < 0.001). Activated endothelial IL-1beta, IL-6, and IL-8 concentrations and ICAM-1 expression are attenuated only by lidocaine at concentrations larger than clinically relevant concentrations.     

Effect of midazolam on in vitro cerebral endothelial ICAM-1 expression induced by astrocyte-conditioned medium

BACKGROUND AND OBJECTIVE: Astrocytes exposed to hypoxia produce pro-inflammatory cytokines and upregulate intercellular adhesion molecule-1 on cerebral endothelium. This study investigated the effects of midazolam on this response. METHODS: Mouse astrocytes were exposed to 4 h of hypoxia and 24 h of reoxygenation. Astrocyte-conditioned medium were applied to mouse cerebral endothelial cell cultures for 4 h and 24 h in normoxia. Endothelial cells were pre-incubated for 1 h with midazolam (0, 5, 50 microg L-1). Flow cytometry was used to estimate endothelial ICAM-1 expression. IL-1beta concentrations were measured with ELISA. Repeated comparisons were made using ANOVA and post hoc Tukey Test as appropriate. Data are mean (SD). RESULTS: Mouse cerebral endothelial cell ICAM-1 expression was greater after 24 h exposure to hypoxia-reoxygenation astrocyte-conditioned medium compared to normoxic astrocyte-conditioned medium (mean channel flouresence 112.5 (9.5) vs. 81.5 (7.5), P = 0.01). ICAM-1 expression was decreased by midazolam (5 microg L(-1)) compared to control (mean channel flouresence 81.35 (7.5) vs. 112.5 (9.5), P = 0.01). Supernatant IL-1beta concentrations (pg mL(1)) in astrocytes exposed to hypoxia-reoxygenation were greater than those exposed to normoxia (16.41 (2.35) vs. 10.5 (2.13), P = 0.01). CONCLUSION: We conclude that decreased cerebral endothelial ICAM-1 expression in response to activated glial cell compartment by midazolam may decrease post ischaemic brain inflammation and secondary brain injury.     

The effect of lidocaine on in vitro neutrophil and endothelial adhesion molecule expression induced by plasma obtained during tourniquet-induced ischaemia and reperfusion

BACKGROUND: Changes in neutrophil and endothelial adhesion molecule expression occur during perioperative ischaemia and reperfusion (I/R) injury. We investigated the effects of lidocaine on neutrophil-independent changes in neutrophil and endothelial adhesion molecule expression associated with tourniquet-induced I/R. METHODS: Plasma was obtained from venous blood samples (tourniquet arm) taken before (baseline), during, 15 min, 2 and 24 h following tourniquet release in seven patients undergoing elective upper limb surgery with tourniquet application. Isolated neutrophils from healthy volunteers (n = 7) were pretreated in the presence or absence of lidocaine (0.005, 0.05 and 0.5 mg mL(-1) for 1 h, and then incubated with I/R plasma for 2 h. Human umbilical vein endothelial cells (HUVECs) were pretreated in the presence or absence of lidocaine (0.005, 0.05 and 0.5 mg mL(-1)) for 1 h, and then incubated with the plasma for 4 h. Adhesion molecule expression was estimated using flow cytometry. Data were analysed using ANOVA and post hoc Student-Newman-Keuls tests. RESULTS: I/R plasma (withdrawn 15 min following tourniquet release) increased isolated neutrophil CD11b (P = 0.03), CD18 (P = 0.01) and endothelial intercellular adhesion molecule-1 (ICAM-1) (P = 0.008) expression compared to baseline. CD11b, CD18 and ICAM-1 expression on lidocaine (0.005 mg mL(-1)) treated neutrophils was similar to control. CD11b (P < 0.001), CD18 (P = 0.03) and ICAM-1 (P = 0.002) expression on lidocaine (0.05 mg mL(-1)) treated neutrophils and HUVECs was less than that on controls. CONCLUSION: Increased in vitro neutrophil and endothelial cell adhesion molecule expression on exposure to plasma obtained during the early reperfusion phase is diminished by lidocaine at greater than clinically relevant plasma concentrations.     

Effect of aprotinin on in vitro cerebral endothelial ICAM-1 expression induced by astrocyte-conditioned medium

BACKGROUND AND OBJECTIVE: Aprotinin administration may decrease the incidence of stroke associated with coronary artery bypass surgery by an unknown mechanism. Astrocytes exposed to hypoxia produce proinflammatory cytokines and upregulate intercellular adhesion molecule (ICAM)-1 on cerebral endothelium. This study investigated the effects of aprotinin on cerebral endothelial activation by hypoxic astrocytes in vitro. METHODS: Mouse astrocytes were exposed to hypoxia in an anaerobic chamber for 4 h followed by reoxygenation for 24 h. Astrocyte-conditioned medium (ACM) collected from mouse astrocytes subjected to hypoxia/reoxygenation (HR) or normoxia were applied to mouse cerebral endothelial cell (MCEC) cultures for 4 and 24 h in normoxia. Endothelial cells were preincubated for 1 h with aprotinin (1600 KIU mL(-1)) prior to exposure to ACM. Flow cytometry was used to estimate endothelial ICAM-1 expression. Interleukin (IL)-1beta space concentrations in ACM were estimated with enzyme-linked immunosorbent assay (ELISA). Repeated comparisons were made using analysis of variance (ANOVA) and post hoc Tukey test as appropriate. P < 0.05 was considered significant. Data is presented as mean (standard deviation, SD). RESULTS: MCEC ICAM-1 expression was greater after 24 h exposure to HR-ACM compared to normoxic-ACM (mean channel flouresence (MCF) 107.5 (4.5) vs. 74.3 (4.5), respectively, P < 0.001). ICAM-1 expression was decreased by aprotinin preincubation compared to control (MCF 91.0 (1.1) vs. 107.5 (2.1), P = 0.006). Supernatant IL-1beta concentrations in astrocytes exposed to HR were greater than those exposed to normoxia (7.1 (0.2) vs. 4.1 (0.2), P = 0.01). CONCLUSIONS: This may be a neuroprotective mechanism associated with aprotinin administration.     

Effect of beta2-adrenergic receptor agonists on intercellular adhesion molecule (ICAM)-1, B7, and CD40 expression in mixed lymphocyte reaction

BACKGROUND: The plasma interleukin (IL)-18 level is elevated in acute rejection after organ transplantation. Although beta2-adrenergic receptor (AR) agonists suppress the rejection of organ and tissue transplants, little is known about their action mechanisms. We examined the effects of endogenous catecholamines and beta2-AR agonists on the expression of intercellular adhesion molecule (ICAM)-1, B7.1, B7.2, CD40, and CD40 ligand (CD40L) in human mixed lymphocyte reaction (MLR) and in an in vitro model of acute rejection in the presence or absence of IL-18. METHODS: ICAM-1, B7.1 B7.2, CD40, and CD40L expression on monocytes was measured by flow cytometry, and the production of interferon (IFN)-gamma and IL-12 was determined by enzyme-linked immunosorbent assay. Lymphocytes proliferation in MLR was measured by [3H]-thymidine uptake. The relevant AR subtypes were characterized using subtype-selective agonists and antagonists. RESULTS: beta2-AR agonists inhibited the expression of ICAM-1 and CD40 during MLR in the absence of IL-18. Among IL-18-induced expression of ICAM-1, B7.1, B7.2, CD40, and CD40L, beta2-AR agonists inhibited ICAM-1 and CD40 expression. beta2-AR agonists prevented the production of IFN-gamma and IL-12 in the presence of IL-18 but had no effect in the absence of IL-18. beta2-AR agonists inhibited lymphocyte proliferation in IL-18-treated MLR. CONCLUSIONS: We found that beta2-AR agonists strongly inhibited the expression of ICAM-1 and CD40, irrespective of the presence or absence of IL-18, which is different from that of histamine and prostaglandin E2.     

Ketamine does not inhibit inflammatory responses of cultured human endothelial cells but reduces chemotactic activation of neutrophils

BACKGROUND: Ketamine is a widely used general anaesthetic, which has been reported to inhibit neutrophil function and neutrophil-endothelial interaction. To date, however, it is unknown whether ketamine has any direct effects on endothelial cells with respect to inflammation. Therefore, we investigated the influence of varying concentrations of ketamine (0.5, 1, and 3 microM) on the endothelial expression of cytokines and adhesion molecules with relevance for inflammation. METHODS: Cultured human umbilical vein endothelial cells were stimulated with tumor necrosis factor alpha (TNFalpha, 2.5 ng/ml) for 4 h in the absence or presence of ketamine. The adhesion molecules ICAM-1 and E-selectin on the endothelial cells were measured by flow cytometry. Release of the proinflammatory cytokines IL-6 and IL-8 by endothelial cells was quantified by ELISA. The acute effect of ketamine on leukocyte activation by the supernatant of endothelial cells pre-stimulated with TNFalpha (4 h) was tested by flow cytometric measurement of CD11b, a leukocyte activation marker, after 15 min of coincubation. RESULTS: TNFalpha caused dramatic upregulation of both adhesion molecules (15-fold and 5-fold vs. control for ELAM-1 and ICAM-1, respectively) and of both cytokines (500-fold and 1.8-fold for IL-6 and IL-8, respectively). No concentration of ketamine employed in our study had any effect on these inflammatory parameters. However, activation of leukocytes by supernatant of TNFalpha-conditioned endothelial cells (70% increase of CD11b) was attenuated by coincubation of the PMN with 0.5 and 5 microM ketamine (47% and 44% increase, respectively). CONCLUSION: These data suggest that ketamine exerts its antiinflammatory actions primarily via inhibition of leukocyte reactivity. Indeed, no inhibition of endothelial responses was detectable in our study.     

Markers of inflammation and atherosclerosis in Egyptian patients with systemic lupus erythematosus

BACKGROUND: Cardiovascular events are markedly increased in systemic lupus erythematosus (SLE) and the mechanism of atherogenesis remains poorly understood. Low-grade inflammation and endothelial dysfunction play pivotal roles in the initiation, progression and propagation of the atherosclerotic process. Several methods have been employed to assess endothelial function, among them the measurement of biomarkers of endothelial activation and dysfunction (intercellular adhesion molecule (ICAM)-1). Since then, it has been reported that such biomarkers play a more important role than traditional risk factors in cardiovascular disease. OBJECTIVE: To measure (tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and ICAM-1) levels as markers of inflammation and atherosclerosis in 40 Egyptian patients with SLE with various degrees of activity in comparison with 20 healthy volunteers, and to investigate their relationship to disease activity and hypertension. METHODS: Sixty subject (40 with SLE and 20 healthy controls) were the subject of this study, their clinical disease activity was scored according to the SLE Disease Activity Index (SLEDAI), and serum sampling was obtained for TNF-alpha, IL-6 and ICAM-1 level assay. Renal biopsy was carried out and examined by light microscopy. The mean level of TNF-alpha, IL-6 and ICAM-1 were significantly higher in SLE patients with active disease (766.95 +/- 357.82 Pg/mL, 135.4 +/- 54.23 Pg/mL, 826.05 +/- 367.1 Pg/mL) when compared with those with inactive disease (314.01 +/- 100.87 Pg/mL, 47.33 +/- 18.61 pg/mL, 441.33 +/- 225.19 Pg/mL) and healthy control volunteers (172.7 +/- 39.19 Pg/mL, 21.15 +/- 10.99 Pg/mL, 111.5 +/- 17.36 Pg/mL), respectively. Furthermore, these levels were significantly higher in hypertensive (614.08 +/- 333.05 Pg/mL, 107.86 +/- 54.96 Pg/mL and 862.13 +/- 333.29 Pg/mL) compared to normotensive patients (267.5 +/- 112.72 Pg/mL, P = 0.008, 35.75 +/- 20.26 Pg/mL, P = 0.02I, and 337.25 +/- 235.62 Pg/mL, P = 0.02) for TNF-alpha, IL-6 and ICAM, respectively. There were no statistically significant difference regarding age, sex, smoking, cholesterol and high-density lipoprotein (HDL) levels between hypertensive and normotensive patients. CONCLUSION: A high concentration of soluble ICAM-1 in Egyptian patients with SLE and nephritis is reported here for the first time. Our finding of increased concentrations of TNF-alpha, IL-6 and ICAM-1 in Egyptian patients with SLE and lupus nephritis underlines the importance of inflammation and endothelial involvement in this disorder, but their predictive value in the disease monitoring needs to be further studied.     

The influence of propofol on the expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in reoxygenated human umbilical vein endothelial cells

BACKGROUND: Leucocytes are a pivotal component of the inflammatory cascade that results in tissue injury in a large group of disorders. Free radical production and endothelial activation promote leucocyte-endothelium interactions via endothelial expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) which augment these processes, particularly in the setting of reperfusion injury. Propofol has antioxidant properties which may attenuate the increased expression of these molecules that is observed. METHODS: Cultured human umbilical vein endothelial cells were exposed to 20 h of hypoxia, then returned to normoxic conditions. Cells were treated with saline, Diprivan 5 microg mL(-1) or propofol 5 microg mL(-1), for 4 h after reoxygenation and were examined for ICAM-1 and VCAM-1 expression. RESULTS: Hypoxia did not increase the expression of ICAM-1/VCAM-1. ICAM-1 expression peaked 12 h after reoxygenation (21.75(0.6) vs. 9.6(1.3), P = 0.02). Propofol, but not Diprivan, prevented this increase (8.2(2.9) vs. 21.75(0.6), P = 0.009). VCAM-1 expression peaked 24 h after reoxygenation (9.8(0.9) vs. 6.6(0.6), P = 0.03). Propofol and Diprivan prevented this increase, with no difference between the two treatments observed (4.3(0.3) and 6.4(0.5) vs. 9.8(0.9), P = 0.001, 0.02, respectively). CONCLUSION: These effects are likely to be attributable to the antioxidant properties of propofol, and suggest that propofol may have a protective role in disorders where free radical mediated injury promotes leucocyte-endothelium adhesive interactions.     

Involvement of endothelial cell adhesion molecules in the development of anti-Thy-1 nephritis

To study an involvement of glomerular endothelial cells in the development of anti-Thy-1 nephritis, we examined the expression of endothelial cell adhesion molecules during the course of this model. Ribonuclease protection assay elucidated that expression of mRNA for intercellular adhesion molecule-1 (ICAM-1) was markedly enhanced in the glomeruli with a peak at 2 h (6.5-fold, p < 0.05) after the anti-Thy-1 antibody injection when mesangial cell lysis was recognized and IL-1beta mRNA expression was induced in the glomeruli. The glomerular ICAM-1 was predominantly localized in the endothelial cells and was intensely immunostained at day 1 in the glomerular endothelial cells. In contrast, platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial-cadherin mRNA expression increased gradually with a peak at day 6 (2.6-fold (p < 0.05) and 4.2-fold (p < 0.05), respectively) in the glomeruli with mesangial proliferative lesion. PECAM-1 was also immunolocalized in the glomerular endothelial cells and the immunoreactivity was greatly enhanced at day 6. Glomerular expression of vascular cell adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 (E-selectin) was unchanged at a low level during the course of anti-Thy-1 nephritis. Blocking of ICAM-1 by administration of anti-ICAM-1 antibody showed significant decrease in the number of polymorphonuclear leukocytes accumulating in the glomeruli by 45.7% (9.4 +/- 0.2 vs. 5.1 +/- 0.1 per glomerular cross section, p < 0.01) at 2 h. These results suggest a significant involvement of glomerular endothelial cells in the development and repair of anti-Thy-1 nephritis via direct or indirect intercellular interactions between mesangial cells and glomerular endothelial cells.     

Interleukin-1beta and interleukin-6 stimulate matrix metalloproteinase-9 secretion in cultured myenteric glia

Western blotting of culture media of myenteric glia stained positive for GFAP revealed increased secretion of matrix metalloproteinase-9 (MMP-9) in interleukin-1beta (IL-1beta) stimulated cells (10 ng/mL) versus control (142 +/- 19 versus 42 +/- 19, P < 0.05). Interleukin-6 (IL-6) stimulated cells also showed increased expression of MMP-9 (10 ng/mL) versus control (69 +/- 14 versus 4 +/- 2, P < 0.01). Control and cytokine-stimulated cells secreted MMP-2 constituitively. Gelatin zymography demonstrated that products were biologically active. Cytoplasmic staining for MMP-9 was detected in IL-1beta and IL-6-stimulated cells but was negligible in controls. Cultured myenteric glia are responsive to IL-1beta and IL-6 stimulation by secreting MMPs into the extracellular environment.     

TGF-beta(1) down-regulates inflammatory cytokine-induced VCAM-1 expression in cultured human glomerular endothelial cells.

BACKGROUND: Endothelial cells are active participants in the processes controlling coagulation, inflammation and the immune response. Variations are recognized between endothelia isolated from different vascular beds as well as from different species. Though transforming growth factor-beta(1) (TGF-beta(1)) has been known to have an anti-inflammatory action, little is known about its effect on expression of cellular adhesion molecules during the inflammatory process in human glomerular endothelial cells. The aim of this study was to determine the effect of TGF-beta(1) on the inflammatory cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human glomerular endothelial cells. METHODS: The culture of human glomerular endothelial cells was established using the normal portion of nephrectomized renal tissues and identified by factor VIII staining and cellular uptake of fluorescent-labelled acetylated low-density lipoprotein (LDL). The endothelial cells were stimulated by interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) with or without TGF-beta(1). Cellular expression of VCAM-1 was measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and VCAM-1 mRNA was measured by Northern blot analysis. RESULTS:TGF-beta(1) (1, 10 and 25 ng/ml) blunted IL-1beta- (5 ng/ml) induced VCAM-1 expression significantly (OD=1.08+/-0.14, 1. 10+/-1.16 and 1.05+/-0.14 vs IL-1beta=1.97+/-0.29, n=6, P<0.05) in ELISA. The addition of TGF-beta(1) (1, 10 and 25 ng/ml) also suppressed TNF-alpha- (10 ng/ml) induced VCAM-1 expression (OD=1. 14+/-0.15, 1.17+/-0.17 and 1.18+/-0.16 vs TNF-alpha=1.96+/-0.26, n=6, P<0.05). The same results were obtained by flow cytometry. TGF-beta(1) (10 ng/ml) inhibited both IL-1beta- (5 ng/ml) and TNF-alpha-(10 ng/ml) induced expression of VCAM-1 (MFI: IL-1beta=90. 8+/- 17.6, IL-1beta+TGF-beta(1)=37.8+/-14.9, TNF-alpha=113.6+/- 12.4, TNF-alpha+TGF-beta(1)=64.3+/-13.8, mean+/-SD, n=3, P<0.05). By Northern blot analysis, TGF-beta(1) (10 ng/ml) significantly suppressed the stimulatory effect of IL-1beta and TNF-alpha. CONCLUSIONS: These results show that TGF-beta(1) down-regulates the inflammatory cytokine-induced expression of VCAM-1 in human glomerular endothelial cells, which could be a novel mechanism for the anti-inflammatory action of TGF-beta(1) during the inflammatory processes in human glomerular diseases.     

Critical involvement of stress-activated mitogen-activated protein kinases in the regulation of intracellular adhesion molecule-1 in serosal fibroblasts isolated from patients with Crohn's disease

BACKGROUND: Stricture formation in Crohn's disease occurs as a result of persistent fibroblast activation. Chronic inflammation seen in patients with Crohn's disease leads to enhanced adhesion molecule expression in fibroblasts. Stress-activated mitogen-activated protein kinases are critical signaling pathways that control expression of intracellular adhesion molecule-1 (ICAM-1) in inflammation. The purpose of this study was to investigate the involvement of stress-activated mitogen-activated protein kinases in the regulation of ICAM-1 expression by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in serosal fibroblasts isolated from patients with Crohn's disease. STUDY DESIGN: Fibroblasts were isolated from serosal biopsies of strictures in patients with Crohn's disease and normal colon in patients with colorectal carcinoma. Cell surface and whole cell ICAM-1 expression were evaluated by flow cytometry and Western blot analysis, respectively. Cells were stimulated with TNF-alpha and IL-1beta. To determine the mitogen-activated protein kinase signaling pathway required for ICAM-1 induction, cells were pretreated with inhibitors to Jun N-terminal kinase, p38 kinase, and p42/44 kinase. RESULTS: Baseline ICAM-1 expression was higher (p < 0.001) in fibroblasts isolated from strictures in patients with Crohn's disease (3.2 +/- 0.3) as compared with nonstrictured Crohn's fibroblasts (2.1 +/- 0.3) and control fibroblasts (1.6 +/- 0.1). TNF-alpha and IL-1beta increased ICAM-1 expression in both control and Crohn's disease. Pretreatment of fibroblasts with the Jun N-terminal kinase inhibitor dimethylaminopurine abolished TNF-alpha- and IL-1beta-stimulated ICAM-1 expression. CONCLUSIONS: Serosal fibroblasts isolated from strictures of patients with Crohn's disease demonstrate enhanced expression of ICAM-1. TNF-alpha and IL-1beta upregulate ICAM-1 expression in serosal fibroblasts through a Jun N-terminal kinase signaling pathway. Specific inhibition of inflammatory signaling pathways could provide novel therapeutic targets for treatment of Crohn's disease.     

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.     

Cytokine regulation of ICAM-1 expression on human renal tubular epithelial cells in vitro.

Regulation of the intercellular adhesion molecule-1 (ICAM-1) expression on human renal tubular epithelial cells in culture (hKEC-1) was investigated. A large proportion of hKEC-1 cells from the primary cultures expressed the ICAM-1 antigen. Supernatants from mixed lymphocyte reaction (MLR) of both specific and third-party combinations augmented the expression of the ICAM-1 antigen, in a dose-dependent manner. A kinetic study revealed maximal augmentation by MLR supernatant on the first day, with a gradual decrease thereafter. Among several recombinant human cytokines tested, i.e., interferon-gamma, tumor necrosis factor-alpha, interleukin 1 alpha and beta, and IL-4, IFN-gamma, TNF-alpha, and IL-1 alpha/beta were shown to augment the expression of ICAM-1. MLR supernatants and IFN-gamma were more effective in augmenting ICAM expression than TNF-alpha and IL-1 alpha/beta. IFN-gamma upregulated ICAM-1 expression in a dose-dependent manner, and maximal augmentation was achieved on the first day. The MLR supernatants were shown to contain IFN-gamma and TNF-alpha, and the activity of the MLR supernatant was partially inhibited by neutralizing antibody against IFN-gamma. These data suggest that cytokines, especially IFN-gamma, TNF-alpha, and IL-1 alpha/beta, released by T cells and antigen-presenting cells upon recognition of alloantigens upregulate ICAM-1 expression on renal tubular epithelial cells. This may result in an increase in the attachment of graft-infiltrating T cells to the renal tubular cells, by the ICAM-1-LFA-1 interaction.     

The number of injections does not influence local anesthetic absorption after paravertebral blockade

PURPOSE: The aims of this study are to determine if the injection of a single large dose of local anesthetics into the paravertebral space increases the risks of inducing toxicity compared with multiple small injections and to describe ropivacaine plasma concentrations resulting from paravertebral blockade. METHODS: Paravertebral blockade was performed using a solution of 10 mL ropivacaine 0.75%, 10 mL lidocaine CO2 2% plus 0.1 mL epinephrine 1:1000 either by a single injection at T3 or T4 (Group S, n = 6) or by five injections of 4 mL each at T2 to T6 (Group M, n = 8). Blood samples were taken at zero, five, ten, 15, 20, 30, 45, 60 and 90 min and at two, three, four, five, six and eight hours. Ropivacaine and lidocaine plasma concentrations were measured by high performance liquid chromatography. RESULTS: Maximal plasma concentrations were comparable for lidocaine: 2.6 +/- 1.3 (S) vs 2.6 +/- 0.8 microg x mL(-1) (M) and for ropivacaine: 1.3 +/- 0.2 (S) vs 1.3 +/- 0.1 microg x mL(-1) (M). Area under the plasma concentration-time curve was higher in Group M for lidocaine: 577.6 +/- 146.1 vs 401.7 +/- 53.2 mg x min(-1) x mL(-1) (P = 0.04) but similar for ropivacaine: 381.1 +/- 95.4 (M) vs 363.1 +/- 85.3 mg x min(-1) x L(-1) (S). CONCLUSIONS: The injection of a single large bolus of local anesthetics into the paravertebral space does not increase its absorption. Maximal ropivacaine plasma concentrations resulting from paravertebral blockade are similar to those reported with equivalent doses of bupivacaine.     

Expression of intercellular adhesion molecule-1 (ICAM-1) during hypoxia-reoxygenation by sinusoidal endothelial cells (SECs) in an obstructive jaundice model

Background/Purpose: The aim of the present study was to investigate the expression of intercellular adhesion molecule-1 (ICAM-1) by sinusoidal endothelial cells (SECs) during hypoxia-reoxygenation in an obstructive jaundice model. Methods: Male Wistar rats aged 6 to 8 weeks were assigned to an obstructive jaundice group (group OJ; n= 8) and a sham-operated control group (group S; n= 8). Using flow cytometry, we determined the percentage of ICAM-1-positive cells and the mean fluorescence intensity (MFI) of ICAM-1 per cell in each group during hypoxia-reoxygenation. Results: The percentage of ICAM-1-positive cells in group OJ did not change significantly under successive incubation conditions. However, the MFI in group OJ differed significantly between normoxia and 24-h renormoxia. The present study investigated the function of isolated SECs from rats with obstructive jaundice and hypoxia-reoxygenation. The percentage of ICAM-1-positive cells and the MFI of ICAM-1-positive cells showed quite similar changes caused by hypoxia-reoxygenation stress in group S and Group OJ. The ICAM-1 expression increased with hypoxia-reoxygenation in both normal and OJ SECs, and the amount of ICAM-1, on the whole, increased. Obstructive jaundice increased the percentage of ICAM-1-positive cells from the normal state. Conclusions: The amount of ICAM-1 increased with hypoxia-reoxygenation in obstructive jaundice than normal state. Received: July 13, 2001 / Accepted: November 16, 2001     

Inflammation in human brain injury: intracerebral concentrations of IL-1alpha, IL-1beta, and their endogenous inhibitor IL-1ra

Following traumatic brain injury (TBI), cascades of inflammatory processes occur. Laboratory studies implicate the cytokines interleukin-1alpha (IL-1alpha) and IL-1beta in the pathophysiology of TBI and cerebral ischemia, whilst exogenous and endogenous interleukin-1 receptor antagonist (IL-1ra) is neuroprotective. We analyzed IL-1alpha, IL-1beta, and IL-1ra in brain microdialysates (100-kDa membrane) in 15 TBI patients. We also analyzed energy-related molecules (glucose, lactate, pyruvate, glutamate, and the lactate/pyruvate ratio) in these brain microdialysates. Mean of mean (+/-SD) in vitro microdialysis percentage recoveries (extraction efficiencies) were IL-1alpha 19.7+/-7.6%, IL-1beta 23.9+/-10.5%, and IL-1ra 20.9+/-6.3%. In the patients' brain microdialysates, mean of mean cytokine concentrations (not corrected for percentage recovery) were IL-1alpha 5.6+/-14.8 pg/mL, IL-1beta 10.4+/-14.7 pg/mL, and IL-1ra 2796+/-2918 pg/mL. IL-1ra was consistently much higher than IL-1alpha and IL-1beta. There were no significant relationships between IL-1 family cytokines and energy-related molecules. There was a significant correlation between increasing IL-1beta and increasing IL-1ra (Spearman r=0.59, p=0.028). There was also a significant relationship between increasing IL-1ra and decreasing intracranial pressure (Spearman r=-0.57, p=0.041). High concentrations of IL-1ra, and also high IL-1ra/IL-1beta ratio, were associated with better outcome (Mann Whitney, p=0.018 and p=0.0201, respectively), within these 15 patients. It is unclear whether these IL-1ra concentrations are sufficient to antagonize the effects of IL-1beta in vivo. This study demonstrates feasibility of our microdialysis methodology in recovering IL-1 family cytokines for assessing their inter-relationships in the injured human brain, and suggests a neuroprotective role for IL-1ra. It remains to be seen whether exogenous IL-1ra or other agents can be used to manipulate cytokine levels in the brain, for potential therapeutic effect.     

RETRACTED ARTICLE: Volume replacement with HES 130/0.4 may reduce the inflammatory response in patients undergoing major abdominal surgery

PURPOSE: To investigate the effects of intravascular volume replacement therapy on the inflammatory response during major surgery. METHODS: Thirty-six patients scheduled for elective abdominal surgery were randomized to receive either 6% hydroxyethylstarch (130,000 Dalton mean molecular weight, degree of substitution 0.4; n = 18, HES-group) or lactated Ringer's solution (RL-group; n = 18) for intravascular volume replacement. Fluid therapy was given perioperatively and continued for 48 hr in the intensive care unit. Volume replacement was guided by physiological parameters. Serum concentrations of interleukin (IL)-6, IL-8 and IL-10 and soluble adhesion molecules (sELAM-1 and sICAM-1) were measured after induction of anesthesia, four hours after the end of surgery, as well as 24 hr and 48 hr postoperatively. RESULTS: Biometric and perioperative data, hemodynamics and oxygenation were similar between groups. On average, 4470 +/- 340 mL of HES 130/0.4 per patient were administered in the HES-group compared to 14310 +/- 750 mL of RL in the RL-group during the study period. Release of pro-inflammatory cytokines IL-6 and IL-8 was significantly lower in the HES-group [(peak values) 47.8 +/- 12.1 pg*dL(-1) of IL-6 and 35.8 +/- 11.2 pg*mL(-1) of IL-8 (HES-group) vs 61.2 +/- 11.2 pg*dL(-1) of IL-6 and 57.9 +/- 9.7 pg*mL(-1) of IL-8 (RL-group); P < 0.05]. Serum concentrations of sICAM-1 were significantly higher in the RL-group [(peak values) 1007 +/- 152 ng*mL(-1) (RL-group) vs 687 +/- 122 ng*mL(-1), (HES group); P < 0.05)]. Values of sELAM-1 were similar in both groups. CONCLUSION: Intravascular volume replacement with HES 130/0.4 may reduce the inflammatory response in patients undergoing major surgery compared to a crystalloid-based volume therapy. We hypothesize that this is most likely due to an improved microcirculation with reduced endothelial activation and less endothelial damage.     

Effect of synthetic protease inhibitor gabexate mesilate on the attenuation of ischemia/reperfusion injury in canine kidney autotransplantation

BACKGROUND: Kidneys from non-heart-beating donors are associated with delayed graft function and a high rejection rate due to the long period of warm ischemia. Gabexate mesilate (GM), a synthetic serine protease inhibitor, has been shown to improve organ function by suppressing cytokine activity and neutrophil function after ischemia/reperfusion. In this study, we evaluated the effect of GM on renal function after warm ischemia in a canine kidney autotransplantation model. METHODS: After 60 minutes of warm ischemia, the left kidney was transplanted into the iliac fossa, and the right kidney was removed. The control group (n = 7) and GM group (n = 7) were evaluated for serum creatinine and blood urea nitrogen (BUN) concentrations, renal tissue blood flow, resistive index, pulsatility index, interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha mRNA expression levels in peripheral blood mononuclear cells, apoptotic index, CD10 immunolabeling as an indicator of brush border injury, and standard histopathology. RESULTS: Compared with controls, administration of GM resulted in lower serum creatinine concentrations (11.3 +/- 2.4 vs 5.2 +/- 3.3 mg/dL at 72 hours; P = .04) and BUN concentrations (188 +/- 26 mg/dL vs 98 +/- 41 mg/dL at 72 hours; P = .04), as well as better tissue blood flow, improvement of brush border injury and apoptotic index (each P < .05). The expression of IL-1beta and TNF-alpha mRNA did not change after administration of GM. CONCLUSIONS: The present study shows that GM protected renal function after warm ischemia/reperfusion by inhibition of serine proteases, maintenance of tissue blood flow, and amelioration of tubular apoptosis.