Impact of Steroids on the Inflammatory Response after Ischemic Acute Kidney Injury in Rats.

Inflammation plays a crucial role in acute kidney injury (AKI). The current study was designed to analyze the influence of prednisolone treatment on the inflammatory reaction during the first 96 h after AKI induction in a rat model. AKI was induced by unilateral clipping of the renal vessels. The treatment group received prednisolone 5 mg/kg s.c. daily. Infiltration rates of macrophages, leukocytes, and T-cells (24, 96 h) as well as plasma concentrations of the inflammatory markers intercellular adhesion molecule, interleukin-1 beta (IL-1ß), IL-18, IL-6, and tumor necrosis factor-alpha (0, 6, 24, 96 h) were determined by fluorescence-activated cell sorting (FACS) analysis only. Ninety-six hours after AKI induction, the prednisolone group demonstrated significantly lower creatinine concentrations compared to the control group (P < 0.05). Twenty-four hours after induction of AKI, a significantly higher rate of infiltrating leukocytes was detectable with FACS analysis in the control group (P < 0.01) with a corresponding significantly higher rate of macrophages after 96 h (P < 0.01). IL-6 and IL-1ß demonstrated a peak after 6 h with a significantly higher release in the control group (IL-6: P < 0.01; IL-1ß: P < 0.05). In contrast to the control group, the prednisolone group demonstrated no further incline of IL-18 after 24 h. The results demonstrate the importance of stretching the observation period in an ischemia-reperfusion-induced AKI setting beyond the first 24 h. Despite the demonstrated protective effects of a continuous prednisolone application, it seems that this single anti-inflammatory agent will not be able to completely suppress the inflammatory response after an ischemia-reperfusion-induced AKI.

Donor Preconditioning After the Onset of Brain Death With Dopamine Derivate n-Octanoyl Dopamine Improves Early Posttransplant Graft Function in the Rat.

Heart transplantation is the therapy of choice for end-stage heart failure. However, hemodynamic instability, which has been demonstrated in brain-dead donors (BDD), could also affect the posttransplant graft function. We tested the hypothesis that treatment of the BDD with the dopamine derivate n-octanoyl-dopamine (NOD) improves donor cardiac and graft function after transplantation. Donor rats were given a continuous intravenous infusion of either NOD (0.882 mg/kg/h, BDD+NOD, n = 6) or a physiological saline vehicle (BDD, n = 9) for 5 h after the induction of brain death by inflation of a subdural balloon catheter. Controls were sham-operated (n = 9). In BDD, decreased left-ventricular contractility (ejection fraction; maximum rate of rise of left-ventricular pressure; preload recruitable stroke work), relaxation (maximum rate of fall of left-ventricular pressure; Tau), and increased end-diastolic stiffness were significantly improved after the NOD treatment. Following the transplantation, the NOD-treatment of BDD improved impaired systolic function and ventricular relaxation. Additionally, after transplantation increased interleukin-6, tumor necrosis factor TNF-α, NF-kappaB-p65, and nuclear factor (NF)-kappaB-p105 gene expression, and increased caspase-3, TNF-α and NF-kappaB protein expression could be significantly downregulated by the NOD treatment compared to BDD. BDD postconditioning with NOD through downregulation of the pro-apoptotic factor caspase-3, pro-inflammatory cytokines, and NF-kappaB may protect the heart against the myocardial injuries associated with brain death and ischemia/reperfusion.

Dopamine treatment of brain-dead Fisher rats improves renal histology but not early renal function in Lewis recipients after prolonged static cold storage.

BACKGROUND: Brain death (BD) and cold preservation are major risk factors for an unfavorable transplantation outcome. Although donor dopamine treatment in brain-dead rats improves renal function and histology in allogeneic recipients, it remains to be assessed if this also holds true for the combinations of BD and prolonged static cold preservation. METHODS: BD was induced in F344 donor rats, which were subsequently treated with NaCl 1 mL/h (BD, n = 11), NaCl/hydroxy ethyl starch (BD-norm, n = 10), or 10 µg/min/kg dopamine (BD-dopa, n = 10). Renal grafts were harvested 4 h after BD and transplanted into bilateral nephrectomized Lewis recipients 6 h after cold preservation in University of Wisconsin solution. Renal function was evaluated by use of serum creatinine and urea concentrations at days 0, 1, 3, 5, and 10. Ten days after transplantation, recipients were killed and the renal allografts were processed for light microscopy and immune histology. RESULTS: Serum urea concentrations at days 5 and 10 were significantly lower in recipients that received a renal graft from dopamine-treated rats; for serum creatinine, only a trend was observed at day 10. Immune histology revealed a lower degree of ED1-positive cells in the donor dopamine-treated group. Under light microscopy, Banff classification revealed significantly less intimal arteritis in these grafts (P < .05). CONCLUSIONS: Although donor dopamine treatment clearly improves renal histology in this model, the beneficial effect on early renal function was marginal. It remains to be assessed if donor dopamine treatment has a beneficial effect on renal function in long-term follow-up.

Different design of enzyme-triggered CO-releasing molecules (ET-CORMs) reveals quantitative differences in biological activities in terms of toxicity and inflammation.

Acyloxydiene-Fe(CO)3 complexes can act as enzyme-triggered CO-releasing molecules (ET-CORMs). Their biological activity strongly depends on the mother compound from which they are derived, i.e. cyclohexenone or cyclohexanedione, and on the position of the ester functionality they harbour. The present study addresses if the latter characteristic affects CO release, if cytotoxicity of ET-CORMs is mediated through iron release or inhibition of cell respiration and to what extent cyclohexenone and cyclohexanedione derived ET-CORMs differ in their ability to counteract TNF-α mediated inflammation. Irrespective of the formulation (DMSO or cyclodextrin), toxicity in HUVEC was significantly higher for ET-CORMs bearing the ester functionality at the outer (rac-4), as compared to the inner (rac-1) position of the cyclohexenone moiety. This was paralleled by an increased CO release from the former ET-CORM. Toxicity was not mediated via iron as EC50 values for rac-4 were significantly lower than for FeCl2 or FeCl3 and were not influenced by iron chelation. ATP depletion preceded toxicity suggesting impaired cell respiration as putative cause for cell death. In long-term HUVEC cultures inhibition of VCAM-1 expression by rac-1 waned in time, while for the cyclohexanedione derived rac-8 inhibition seems to increase. NFκB was inhibited by both rac-1 and rac-8 independent of IκBα degradation. Both ET-CORMs activated Nrf-2 and consequently induced the expression of HO-1. This study further provides a rational framework for designing acyloxydiene-Fe(CO)3 complexes as ET-CORMs with differential CO release and biological activities. We also provide a better understanding of how these complexes affect cell-biology in mechanistic terms.

Enzyme-triggered CO-releasing molecules (ET-CORMs): evaluation of biological activity in relation to their structure.

Acyloxydiene-Fe(CO)3 complexes act as enzyme-triggered CO-releasing molecules (ET-CORMs) and can deliver CO intracellularly via esterase-mediated hydrolysis. The protective properties of structurally different ET-CORMs on hypothermic preservation damage and their ability to inhibit VCAM-1 expression were tested on cultured human umbilical vein endothelial cells (HUVEC) and renal proximal tubular epithelial cells (PTEC) using a structure-activity approach. Cytotoxicity of ET-CORMs, protection against hypothermic preservation damage, and inhibition of VCAM-1 expression were assessed. Cytotoxicity of 2-cyclohexenone and 1,3-cyclohexanedione-derived ET-CORMs was more pronounced in HUVEC compared to PTEC and was dependent on the position and type of the ester (acyloxy) substituent(s) (acetate>pivalate>palmitate). Protection against hypothermic preservation injury was only observed for 2-cyclohexenone-derived ET-CORMs and was not mediated by the ET-CORM decomposition product 2-cyclohexenone itself. Structural requirements for protection by these ET-CORMs were different for HUVEC and PTEC. Protection was affected by the nature of the ester functionality in both cell lines. VCAM-1 expression was inhibited by both 2-cyclohexenone- and 1,3-cyclohexanedione-derived ET-CORMs. 2-Cyclohexenone, but not 1,3-cyclohexanedione, also inhibited VCAM-1 expression. We demonstrate that structural alterations of ET-CORMs significantly affect their biological activity. Our data also indicate that different ET-CORMs behave differently in various cell types (epithelial vs endothelial). These findings warrant further studies not only to elucidate the structure-activity relation of ET-CORMs in mechanistic terms but also to assess if structural optimization will yield ET-CORMs with restricted cell specificity.

Inhibition of neutrophil-mediated production of reactive oxygen species (ROS) by endothelial cells is not impaired in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis patients.

Leucocyte transendothelial migration is strictly regulated to prevent undesired inflammation and collateral damage of endothelial cells by activated neutrophils/monocytes. We hypothesized that in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated vasculitis (AAV) patients' dysregulation of this process might underlie vascular inflammation. Peripheral blood mononuclear cells (PBMC) and neutrophils from AAV patients (n = 12) and healthy controls (HC, n = 12) were isolated. The influence of human umbilical vein endothelial cells (HUVEC) on neutrophil/monocytes function was tested by N-formyl-methionyl-leucyl-phenyl-alanine (fMLP)- and phorbol 12-myristate 13-acetate (PMA)-mediated ROS production, degranulation and interleukin (IL)-8 production. In addition, the ability of lipopolysaccharide (LPS)-stimulated PBMC to produce tumour necrosis factor (TNF)-alpha in the presence or absence of HUVEC was tested. HUVEC inhibited ROS production dose-dependently by fMLP-stimulated neutrophils but did not influence degranulation. No differences between neutrophils from HC and AAV were found. However, in only one active patient was degranulation inhibited significantly by HUVEC only before cyclophosphamide treatment, but not 6 weeks later. Co-cultures of HUVEC with LPS-stimulated neutrophils/monocytes increased IL-8 production while TNF-alpha production was inhibited significantly. There was no apparent difference between AAV patients and HC in this respect. Our findings demonstrate that HUVEC are able to inhibit ROS and modulate cytokine production upon stimulation of neutrophils or monocytes. Our data do not support the hypothesis that endothelial cells inhibit ROS production of neutrophils from AAV patients inadequately. Impaired neutrophil degranulation may exist in active patients, but this finding needs to be confirmed.

Modulation of brain dead induced inflammation by vagus nerve stimulation.

Because the vagus nerve is implicated in control of inflammation, we investigated if brain death (BD) causes impairment of the parasympathetic nervous system, thereby contributing to inflammation. BD was induced in rats. Anaesthetised ventilated rats (NBD) served as control. Heart rate variability (HRV) was assessed by ECG. The vagus nerve was electrically stimulated (BD + STIM) during BD. Intestine, kidney, heart and liver were recovered after 6 hours. Affymetrix chip-analysis was performed on intestinal RNA. Quantitative PCR was performed on all organs. Serum was collected to assess TNFalpha concentrations. Renal transplantations were performed to address the influence of vagus nerve stimulation on graft outcome. HRV was significantly lower in BD animals. Vagus nerve stimulation inhibited the increase in serum TNFalpha concentrations and resulted in down-regulation of a multiplicity of pro-inflammatory genes in intestinal tissue. In renal tissue vagal stimulation significantly decreased the expression of E-selectin, IL1beta and ITGA6. Renal function was significantly better in recipients that received a graft from a BD + STIM donor. Our study demonstrates impairment of the parasympathetic nervous system during BD and inhibition of serum TNFalpha through vagal stimulation. Vagus nerve stimulation variably affected gene expression in donor organs and improved renal function in recipients.

The carbon monoxide releasing molecule (CORM-3) inhibits expression of vascular cell adhesion molecule-1 and E-selectin independently of haem oxygenase-1 expression.

BACKGROUND AND PURPOSE: Although carbon monoxide (CO) can modulate inflammatory processes, the influence of CO on adhesion molecules is less clear. This might be due to the limited amount of CO generated by haem degradation. We therefore tested the ability of a CO releasing molecule (CORM-3), used in supra-physiological concentrations, to modulate the expression of vascular cell adhesion molecule (VCAM)-1 and E-selectin on endothelial cells and the mechanism(s) involved. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were stimulated with tumour necrosis factor (TNF)-alpha in the presence or absence of CORM-3. The influence of CORM-3 on VCAM-1 and E-selectin expression and the nuclear factor (NF)-kappaB pathway was assessed by flow cytometry, Western blotting and electrophoretic mobility shift assay. KEY RESULTS: CORM-3 inhibited the expression of VCAM-1 and E-selectin on TNF-alpha-stimulated HUVEC. VCAM-1 expression was also inhibited when CORM-3 was added 24 h after TNF-alpha stimulation or when TNF-alpha was removed. This was paralleled by deactivation of NF-kappaB and a reduction in VCAM-1 mRNA. Although TNF-alpha removal was more effective in this regard, VCAM-1 protein was down-regulated more rapidly when CORM-3 was added. CORM-3 induced haem oxygenase-1 (HO-1) in a dose- and time-dependent manner, mediated by the transcription factor, Nrf2. CORM-3 was still able to down-regulate VCAM-1 expression in HUVEC transfected with siRNA for HO-1 or Nrf2. CONCLUSIONS AND IMPLICATIONS: Down-regulation of VCAM and E-selectin expression induced by CORM-3 was independent of HO-1 up-regulation and was predominantly due to inhibition of sustained NF-kappaB activation.

Rosiglitazone reduces angiotensin II and advanced glycation end product-dependent sustained nuclear factor-kappaB activation in cultured human proximal tubular epithelial cells.

Tubular damage is a major feature in the development of diabetic nephropathy. This study investigates the effects of the thiazolidindione rosiglitazone on angiotensin II and advanced glycation end product-induced tubular activation in human proximal tubular epithelial cells IN VITRO. Angiotensin II and advanced glycation end products, both induced a dose-dependent sustained activation of the redox-sensitive transcription factor, Nuclear Factor KAPPA B (NF-kappaB). Nuclear translocation of NF-kappaB was evident already after one hour and persistent for more than four days. Co-incubation of proximal tubular epithelial cells with rosiglitazone significantly reduced angiotensin II and advanced glycation end product-mediated generation of reactive oxygen species, angiotensin II-dependent advanced glycation end product formation, NF-kappaB activation, and NF-kappaB-dependent pro inflammatory gene expression. Most importantly, rosiglitazone effects on NFkappaB activation were maximal at later time points, indicating that rosiglitazone treatment confers long lasting renoprotective effects.

High mobility group box 1 and adenosine are both released by endothelial cells during hypothermic preservation.

Hypothermic preservation of solid allografts causes profound damage of vascular endothelial cells. This, in turn, might activate innate immunity. In the present study we employed an in vitro model to study to what extent supernatants of damaged endothelial cells are able to activate innate immunity and to study the nature of these signals. The expression of high mobility group box 1 (HMGB1) and adhesion molecules on human umbilical vein endothelial cell was studied by immunofluorescence, fluorescence activated cell sorter and Western blotting. Cytokine production was performed by enzyme-linked immunosorbent assay. HMGB1 expression was lost completely in endothelial cells after hypothermic preservation. This was associated with cell damage as it occurred only in untreated endothelial cell but not in cells rendered resistant to hypothermia-mediated damage by dopamine treatment. Only supernatants from hypothermia susceptible cells up-regulated the expression of interleukin (IL)-8 and adhesion molecules in cultured endothelial cells in an HMGB1-dependent manner. In whole blood assays, both supernatants of hypothermia susceptible and resistant cells inhibited tumour necrosis factor (TNF)-alpha production concomitantly with an increased IL-10 secretion. The activity of the supernatants was already found after 6 h of hypothermic preservation, and paralleled the decrease in intracellular adenosine triphosphate (ATP) levels. Modulation of TNF-alpha and IL-10 production by these supernatants was abrogated completely by prior treatment with adenosine deaminase and was similar to the response of an A2R agonist. Our study demonstrates that both HMGB1 and adenosine are released during hypothermic preservation. While release of HMGB1 is caused by cell damage, release of adenosine seems to be related to ATP hydrolysis, occurring in both susceptible and resistant cells.

Imatinib mesylate, a new kid on the block for the treatment of anti-neutrophil cytoplasmic autoantibodies-associated vasculitis?

Persistent T cell activation is a common finding in anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitis (AAV) patients. Because imatinib, a selective inhibitor of the ABL, ARG, PDGFR and c-KIT tyrosine kinases, inhibits T cell activation, this study was conducted to evaluate the potential use of imatinib for the treatment AAV patients refractory to conventional therapy. In particular, we investigated the inhibition of T cell activation by this drug and its efficacy on activated T cells from anti-neutrophil cytoplasmic autoantibodies (ANCA)-associated systemic vasculitides (AASV) patients. T cell stimulation has been induced by anti-CD3/anti-CD28 antibodies or by phorbol myristate acetate (PMA)/ionomycin. T cell proliferation was analysed by tritiumthymidine incorporation. Cell cycle progression was determined by propidium iodide staining using fluorescence activated cell sorter (FACS) analysis and by RNAse protection assay (RPA). Cytokine levels were assessed by enzyme-linked immunosorbent assay. T cell proliferation was inhibited significantly by imatinib, due most probably to cell cycle arrest in the G1-phase. This was paralleled by inhibition in the expression of cyclin-dependent kinases 1 and 2 mRNA. The expression of CD25 in naive and memory T cells was decreased significantly by imatinib in activated T cells. Similarly, conversion from naive to memory T cells after T cell activation was impaired by imatinib. Imatinib did not influence interleukin-2 and tumour necrosis factor-alpha production but increased interferon-gamma production. These observed effects of imatinib were similar in T cells from AASV patients and from healthy individuals. Imatinib might be an alternative therapeutical option for AASV patients refractory to conventional therapy.

In vivo effects of cyclic administration of 15-deoxyspergualin on leucocyte function in patients with Wegener's granulomatosis.

15-Deoxyspergualin (DSG) is an alternative treatment modality for Wegener's granulomatosis (WG) patients refractory to conventional treatment. Nevertheless, it is unclear how DSG modulates disease activity in these patients. This study was conducted to investigate which parameters of adaptive and acquired immunity were influenced during two subsequent cycles of DSG treatment. Emphasis was put upon T cell and monocyte activation, neutrophil function and surface expression of proteinase-3 (PR-3). Anti-CD3/anti-CD28 and interleukin (IL)-15/IL-7-mediated T cell proliferation were assessed by fluorescence activated cell sorter (FACS) analysis using carboxyfluorescein succinimidyl ester (CSFE) labelling. Interferon (IFN)-gamma and IL-10 production were determined in the supernatants of these cultures by enzyme-linked immunosorbent assay. Monocyte activation was assessed in lipopolysaccharide (LPS)-stimulated whole blood, using tumour necrosis factor (TNF)-alpha as read-out. Neutrophil function was determined by measuring oxidative burst, chemotaxis and phagocytosis. T cell activation markers and PR3 expression were measured by FACS. All parameters were determined directly before and after each DSG cycle. Anti-CD3/anti-CD28-mediated T cell proliferation was reduced directly after DSG treatment. Directly before a subsequent cycle of DSG was started, T cell proliferation was increased. Similar findings were observed for IFN-gamma and IL-10 production by T cells. DSG did not influence IL-15/IL-7-mediated T cell proliferation. LPS-mediated TNF-alpha production was also impaired directly after DSG treatment. No influence on T cell activation markers, neutrophil function and surface PR-3 expression was observed in peripheral blood of these patients. Our data demonstrate that DSG influences T cell and monocyte activation in a reversible fashion. Although DSG causes neutropenia in these patients, it does not influence neutrophil function.

Effect of pre-treatment with catecholamines on cold preservation and ischemia/reperfusion-injury in rats.

Treatment of organ donors with catecholamines reduces acute rejection episodes and improves long-term graft survival after renal transplantation. The aim of this study was to investigate the effect of catecholamine pre-treatment on ischemia/reperfusion (I/R)- and cold preservation injury in rat kidneys. I/R-injury was induced by clamping the left kidney vessels for 60 min along with a contralateral nephrectomy. Cold preservation injury was induced by storage of the kidneys for 24 h at +4 degrees Celsius in University of Wisconsin solution, followed by syngeneic transplantation. Rats were pre-treated with either dopamine (DA), dobutamine (DB), or norepinephrine (2, 5, and 10 microg/kg/min, each group) intravenously via an osmotic minipump for 24 h before I/R- and cold preservation injury. Pre-treatment with DA (2 or 5 microg/kg/min) and DB (5 microg/kg/min) improved recovery of renal function after I/R-injury and dose dependently reduced mononuclear and major histocompatibility complex class II-positive cells infiltrating the kidney after I/R-injury. One day after I/R-injury, upregulation of transforming growth factor (TGF)-beta 1 and 2 and phosphorylation of p42/p44 mitogen-activated protein kinases was observed in kidneys of animals treated with DA or DB. DA (5 microg/kg/min) and DB (5 microg/kg/min) pre-treatment reduced endothelial cell damage after 24 h of cold preservation. Only DA pre-treatment improved renal function and reduced renal inflammation after 24 h of cold preservation and syngeneic transplantation. Our results demonstrate a protective effect of pre-treatment with catecholamines on renal inflammation and function after I/R- or cold preservation injury. This could help to explain the potent organoprotective effects of catecholamine pre-treatment observed in human kidney transplantation.

Heterogeneity in lipopolysaccharide responsiveness of endothelial cells identified by gene expression profiling: role of transcription factors.

Interindividual differences of endothelial cells in response to endotoxins might contribute to the diversity in clinical outcome among septic patients. The present study was conducted to test the hypothesis that endothelial cells (EC) with high and low proinflammatory potential exist and to dissect the molecular basis underlying this phenomenon. Thirty human umbilical vein endothelial cell (HUVEC) lines were stimulated for 24 h with lipopolysaccharide (LPS) and screened for interleukin (IL)-8 production. Based on IL-8 production five low and five high producers, tentatively called types I and II responders, respectively, were selected for genome-wide gene expression profiling. From the 74 genes that were modulated by LPS in all type II responders, 33 genes were not influenced in type I responders. Among the 41 genes that were increased in both responders, 17 were expressed significantly stronger in type II responders. Apart from IL-8, significant differences in the expression of proinflammatory related genes between types I and II responders were found for adhesion molecules [intercellular adhesion molecule (ICAM-1), E-selectin)], chemokines [monocyte chemoattractant protein (MCP-1), granulocyte chemotactic protein (GCP-2)], cytokines (IL-6) and the transcription factor CCAAT/enhancer binding protein-delta (C/EBP-delta). Type I responders also displayed a low response towards tumour necrosis factor (TNF)-alpha. In general, maximal activation of nuclear factor (NF)-kappaB was achieved in type I responders at higher concentrations of LPS compared to type II responders. In the present study we demonstrate that LPS-mediated gene expression differs quantitatively and qualitatively in types I and II responders. Our results suggest a pivotal role for common transcription factors as a low inflammatory response was also observed after TNF-alpha stimulation. Further studies are required to elucidate the relevance of these findings in terms of clinical outcome in septic patients.

Abnormalities of CD4 T cell subpopulations in ANCA-associated vasculitis.

In patients with ANCA-associated vasculitis (AAV), CD25 expression is increased on circulating T cells. Although in animal experiments the role of CD4(+) CD25(+) T-regulatory-cells (T(reg)) in protection against autoimmunity is well established, the role of these cells in AAV is unknown. To investigate the hypothesis that an increased expression of CD25 on T cells is related to persistent T cell activation and not to disturbances in T(reg) cells in AAV (34 patients, six of them after renal transplantation), we investigated CD25 expression in different subpopulations of CD4(+) cells and FOXP3 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR). In addition, T cell proliferation and cytokine secretion after stimulation with anti-CD3 and anti-CD28 and intracellular cytokine production after stimulation with phorbol myristate acetate (PMA)-ionomycin was determined. Controls were non-vasculitic renal transplant patients (n = 9) and healthy controls (HC) (n = 13). In AAV the total number of lymphocytes, CD4(+) lymphocytes and the percentage of naive T cells are lower than in HC and RTX. An increased percentage of CD25(+) cells was found in AAV and AAV/RTX, irrespective of disease activity, but not in HC or RTX. This was confined to the naive (CD4(+) CD45RB(high)) population only. FOXP3 mRNA expression in CD4(+) T cells did not differ between AAV patients and healthy controls. In vitro T cell proliferation was enhanced in AAV patients compared to HC (P < 0.01). PBMC of AAV patients produced significantly less interleukin (IL)-10 and interferon (IFN)-gamma after anti-CD3/CD28 stimulation. The percentage of IL-10 and IL-12, but not IFN-gamma, IL-4 or tumour necrosis factor (TNF)-alpha-producing cells was significantly higher in patients compared to HC. These findings were confined to the memory population of CD4(+) cells. We conclude that AAV patients are lymphopenic and have low numbers of CD4(+) T cells, which seem to be in a persistent state of activation.

Fractalkine is not a major chemoattractant for the migration of neutrophils across microvascular endothelium.

Inflammatory responses during sepsis are determined by leucocyte recruitment into inflamed tissues. Both chemokines and adhesion molecules are believed to be involved in this process. As fractalkine exists as transmembrane protein with cell adhesion properties and as soluble chemotactic factor, the present study was conducted to study the role of fractalkine, produced by microvascular and macrovascular endothelial cells, in neutrophil recruitment. Lung microvascular endothelial cells (LMVECs) stimulated with lipopolysaccharide, tumour necrosis factor-alpha or interleukin-1 (IL-1) produced much more fractalkine compared with the macrovascular human umbilical vein endothelial cells (HUVECs). No differences were found between microvascular endothelial cells of different organs. Chemotactic activity in supernatants was significantly stronger in stimulated LMVEC when compared with HUVEC. Although recombinant fractalkine induced migration of neutrophils, IL-8 and monocyte chemoattractant protein-1 were found to be more strictly required. In vivo fractalkine was strongly upregulated in septic lung and kidney. Our data suggest that fractalkine production per se does not explain the preference for inflammation in the lung of septic patients.

Nongenomic effects of aldosterone on human renal cells.

The development of chronic renal insufficiency may be partially mediated by the nongenomic action of aldosterone. Here we investigate whether aldosterone could evoke a nongenomic action in primary cultures of human renal cells. Intracellular Ca(2+) ([Ca(2+)](i)) and cAMP were measured in human mesangial cells (MC), glomerular visceral epithelial cells (GVEC), and proximal and distal tubular epithelial cells (Ptec and Dtec) in the presence of aldosterone (10-100 nmol/liter) by fura-2 fluorescence and RIA, respectively. In MC, Ptec, and Dtec, aldosterone increased [Ca(2+)](i) within 1 min, whereas in GVEC, only a minor effect was found. Preincubation of cells with spironolactone did not blunt this effect. Hydrocortisone, used at a concentration 100-fold higher than that of aldosterone, did not affect [Ca(2+)](i.) In MC, Ptec, and Dtec, a dose-dependent increase ( approximately 1.3- to 1.5-fold) in intracellular cAMP levels was found. These data demonstrate a nongenomic action of aldosterone in human MC, Ptec, and Dtec. As these effects occur at concentrations close to free plasma aldosterone levels in man, they may be of physiological relevance and may contribute to renal injury.

Human proteinase 3 can inhibits LPS-mediated TNF-alpha production through CD14 degradation: lack of influence of antineutrophil cytoplasmic antibodies.

The present study was conducted to investigate if proteinase-3 (PR3) is able to influence lipopolysaccharide (LPS) responses of monocytes via degradation of CD14 and if antineutrophil cytoplasmic antibodies (ANCA) may modify this process. Recombinant (r) CD14 and CD14 expressed on monocytes were investigated for PR3 mediated degradation by SDS-PAGE and FACS analysis, respectively. TNF-alpha production in whole blood was used to determine functional consequences of CD14 degradation. PR3 degraded rCD14 in a dose- and time-dependent fashion. Major degradation products were found with apparent molecular weight of 45, 25 and 10 kDa. Treatment of PR3 with PMSF completely abolished CD14 degradation. ANCA IgG did not inhibit CD14 degradation. In whole blood, addition of PR3 resulted in diminished CD14 expression on monocytes. In contrast, CD14 was increased in a subpopulation of cells that expressed major histocompatibility (MHC) class II and PR3, but lacked expression of CD64 and CD16. LPS mediated TNF-alpha production in whole blood was significantly inhibited when preincubated with PR3. This study demonstrates that PR3 can degrade rCD14 and that PR3 differentially affects CD14 expression in subsets of monocytes. ANCA IgG does not play a significant role herein.

Modulation of chemokine production in lung microvascular endothelial cells by dopamine is mediated via an oxidative mechanism.

Serum concentrations of catecholamines are high in patients with sepsis or acute respiratory distress syndrome (ARDS). Because chemokines mediate the recruitment of neutrophils into inflammatory sites, we addressed the question of whether dopamine (DA) is able to influence chemokine production in endothelial cells under basal and proinflammatory conditions. To this end, lung microvascular endothelial cells (LMVEC) were stimulated or not for 24 h with the bacterial toxins lipopolysaccharide (LPS) (1 microg/ml) or lipoteichonic acid (LTA) (10 microg/ml) in the presence or absence of various concentrations of DA (1-100 microg/ml). Whereas under basal and stimulatory conditions, the addition of DA to endothelial cells dose-dependently increased IL-8 production, the production of ENA-78 and Gro-alpha was significantly inhibited (P < 0.01). This effect could still be demonstrated when the cells were stimulated for up to 3 h with LPS before DA administration. Similar findings were detected for the mRNA expression of these chemokines. The influence of DA on chemokine production was not receptor mediated and could be prevented by antioxidants or radical scavengers. Moreover, addition of H(2)O(2) to endothelial cells gave results similar to those observed with DA stimulation, suggesting a pivotal role for reactive oxygen species in DA-mediated modulation of chemokine production in endothelial cells. Our data thus demonstrate that DA administration results in the induction of oxidative stress, with profound effects on endothelial chemokine production.

ANCA titres, even of IgG subclasses, and soluble CD14 fail to predict relapses in patients with ANCA-associated vasculitis.

BACKGROUND: Antineutrophil cytoplasmic autoantibodies (ANCA) are presumed to reflect disease-activity and to be useful for guidance of immunosuppressive therapy of ANCA-associated systemic vasculitis (AASV), but with respect to conventional ANCA assays this is controversial. ANCA titres, measured in the IgG3 subclass and modern capture ELISAs, have been said to be superior predictors of relapses of AASV. METHODS: In this retrospective study serial measurements of ANCA parameters and soluble CD14 (sCD14) were performed in 169 consecutive sera over a median of 21 months in 18 patients with AASV and related to disease activity, assessed by Birmingham Vasculitis Activity Score (BVAS) for new or deteriorated (BVAS1), and for chronic disease activity (BVAS2). Fourteen patients had Wegener's granulomatosis (WG) and were C-ANCA positive with Pr 3-antibodies and four patients had microscopic polyangiitis (MPA) with P-ANCA and MPO-antibodies. In WG patients ANCA by IIF, Pr 3-ELISA for IgG, IgG1, IgG3, IgG4 and sCD14 were measured, as well as capture ELISA for Pr 3, and in MPA patients ANCA by IIF, MPO-ELISA for IgG and IgG1, IgG3, IgG4, and sCD14 respectively. In eight patients, data collection started at diagnosis, in 10 patients at remission. RESULTS: The parameters predicted neither the nine major relapses (increase of immunosuppression necessary), nor the 26 minor relapses (increase of BVAS1>2) with sufficient sensitivity (>80%) or specificity (> 90%90%), and they also failed to predict relapses within the following 2 months. ANCA-IgG3 and capture ELISA for Pr 3 were not advantageous for prediction of relapses (sensitivity 0.45 and 0.19 respectively), and sCD14 remained elevated in all samples irrespective of disease activity. CONCLUSIONS: There is no rationale for serial measurements of ANCA in AASV. For changes of therapy, the ANCA parameters should only be used in conjunction with clinical information.