Anti-CII mAb transfer
The experimental protocol was approved by the local animal ethics committee and followed the National Institutes of Health guidelines for the care and use of laboratory animals. DBA1/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). All mice were kept under standard conditions at the animal care facility of the University of Rostock.
For the induction of immune-complex-induced inflammation, 14-week-old mice (n = 8 per group) received two mAbs (CIIC1 and M2.139 [5, 15]; 4.5 mg of each) intravenously into the caudal vein. Simultaneously the animals were treated with either 100 or 300 μg of recombinant human ALP [5, 16] (kindly provided by Dr Heinzel-Wieland, Darmstadt, Germany) or 300 μg of a control protein (human serum albumin; Octapharma, Langenfeld, Germany), or received an equal volume of saline only. After 6 and 24 hours, animals (n = 6 to 8 per time point) were anaesthetized with ketamine (90 mg/kg body weight) and xylacin (6 mg/kg) for subsequent in vivo multifluorescence microscopy. Animals were placed on a heating pad to maintain a body temperature of 37°C and a catheter was placed in the left jugular vein for the application of fluorescent dyes. For in vivo multifluorescence microscopy of synovial microcirculation, the knee joint model was used as described . In brief, skin was incised distal to the patella tendon. After removal of the overlying soft tissues, the patella tendon was cut transversely and the proximal and distal parts carefully mobilized. After exposure, the Hoffa's fatty body was superfused with physiological saline solution ay 37°C to prevent tissues from drying and was finally covered with a glass slide. After a 15-minute stabilization period after surgical preparation, in vivo microscopy of the synovial tissue was performed. At the end of each experiment the animals were killed by exsanguination.
Human ALP was produced as a recombinant protein in Escherichia coli and subsequently purified to homogeneity by a multistep purification protocol by ion-exchange, metal-chelate and size-exclusion chromatography as originally described by Heinzel-Wieland and colleagues . Analytical reverse-phase chromatography revealed a single peak, and SDS-PAGE exhibited a single band with the expected electrophoretic mobility on being stained with silver. The material was tested for inhibition of granulocyte elastase and cathepsin G. To ensure the removal of any endotoxin contamination, the ALP charges were run on detoxi-gel columns (polymyxin B-conjugated columns; Perbio Science, Bonn, Germany) in accordance with the manufacturer's instructions. Routine testing of the final ALP preparations before use demonstrated that for all analysed samples the endotoxin content remained below the detection limit (0.05 U/ml) of the applied Limulus amoebocyte lysate gel-clot assay (Sigma, Taufkirchen, Germany).
In vivofluorescence microscopy
After intravenous injection of fluorescein isothiocyanate (FITC)-labelled dextran (15 mg/kg body weight) (Sigma, Deisenhofen, Germany) and rhodamine 6G (0.15 mg/kg body weight) (Sigma), in vivo microscopy was performed with a Zeiss microscope (Axiotech Vario 100HD; Carl Zeiss, Jena, Germany) equipped with a 100 W mercury lamp and filter sets for blue (excitation at 465 to 495 nm; emission at more than 505 nm) and green (excitation at 510 to 560 nm; emission at more than 575 nm) epi-illumination. By the use of water-immersion objectives (× 20 W/numerical aperture (NA) 0.5 and × 40 W/NA 0.8; Zeiss) final magnifications of × 306 and × 630 were achieved. Images were recorded by means of a charge-coupled device video camera (FK 6990-IQ-S; Pieper, Schwerte, Germany) and transferred to an S-VHS video system for subsequent off-line analysis.
For quantitative off-line analysis a computer-assisted microcirculation image analysis system was used (CapImage v.7.4; Zeintl, Heidelberg, Germany). For the assessment of interaction between leukocytes and endothelial cells in postcapillary venules, the flow behavior of leukocytes was analysed with respect to free floating, rolling and adherent leukocytes [17, 18]. Rolling leukocytes were defined as those cells moving along the vessel wall at a velocity less than 40% of that of leukocytes at the centreline and were expressed as a percentage of the total leukocyte flux. Venular leukocyte adherence was defined as the number of leukocytes not moving or detaching from the endothelial lining of the venular vessel wall during an observation period of 20 seconds. Assuming cylindrical microvessel geometry, leukocyte adherence was expressed as non-moving cells per endothelial surface (n/mm2), calculated from the diameter and length of the vessel segment analysed.
Results are given as means ± SEM. After proving the assumption of normality, comparisons between the experimental groups were performed by one-way analysis of variance (ANOVA), followed by the appropriate post-hoc multiple comparison procedure, including Bonferroni correction. Statistical significance was set at p < 0.05.
Isolation of human granulocytes
Blood cells were obtained from heparinized venous blood from healthy donors. In brief, granulocytes were isolated by discontinuous Percoll density gradient centrifugation with 70% and 62% Percoll (Biochrom, Berlin, Germany). The phase containing the granulocytes was separately transferred to a new tube and washed with PBS. Contaminating erythrocytes were removed by hypotonic lysis and a subsequent centrifugation step. The purified granulocytes were recovered from the cell pellet and resuspended in DMEM at a final concentration of 106 cells/ml.
Leukocytes adhesion assay
The leukocyte adhesion assays were performed as described by Hammel and colleagues , with slight modifications. glEND.2 cells were produced and characterized as described for mlEND.1 cells . They were seeded on Labtek chamber slides (6 × 104) and grown to confluence. glEND.2 cells were incubated for 2 hours with different concentrations of ALP (100 nM to 5 μM). The upregulation of adhesion molecules on the glEND.2 cells was induced by activating the cells with IL-1β (20 ng/ml, R&D Systems GmbH, Wiesbaden, Germany) for 4 hours at 37°C. The cells were washed intensively with DMEM and the freshly isolated human granulocytes were added in a volume of 200 μl to the endothelial cell layer and agitated horizontally at 75 r.p.m. for 20 minutes at 4°C. Cells were washed carefully three times in DMEM and fixed overnight in 2.5% glutardialdehyde (Roth, Karlsruhe, Germany). On the next day, the adherent cells were evaluated by direct microscopic counting of four randomly chosen visual fields. Some experiments were performed with the human endothelial cell line EA.hy 926 that was originally derived by the fusion of human umbilical-vein endothelial cells with the epithelial cell line A549 . In that case the granulocytes were prelabelled with carboxyfluorescein diacetate succinimidyl diester (CFDA-SE), Molecular Probes, Eugene, OR, USA), to facilitate the counting of adherent granulocytes in randomly chosen visual fields by fluorescence microscopy (× 10/NA 0.3, objective Axiophot; Zeiss) as described .
E-selectin expression analysis by FACS
glEND.2 cells were seeded on 48-well plates (4 × 104) in complete low-glucose DMEM and grown to 80% confluence. ALP treatment was performed after a PBS washing step with 2 to 12 μM ALP for 2 hours at 37°C. Medium was removed and the cells were washed twice with PBS and activated for 4 hours with 20 ng/ml IL-1β. E-selectin expression was detected on staining with a rat anti-mouse CD62E-biotin (dilution 1:400; BD Biosciences Pharmingen, San Diego, CA, USA) antibody and phycoerythrin-labelled avidin (dilution 1:1,000; BD Biosciences Pharmingen). After a final centrifugation, the cells were resuspended in 1 ml of PBS and their fluorescence was measured by flow cytometry in a Coulter Epics XL flow cytometer (Beckman CoulterCorp., Miami, FL, USA). For the analysis, the gates of forward and sideways light scatter were set on the glEND.2 cell population. The average of E-selectin expression in the endothelial cell population was expressed as the mean of the fluorescence intensity.
Analysis of nuclear NF-κB DNA binding activity
glEND.2 cells were grown to 80% confluence in 75 cm3 culture flasks and treated with different concentrations of ALP (250 nM to 10 μM) for 2 hours at 37°C. After an intensive washing step, cells were activated with 10 ng/ml IL-1β for 1 hour. Subsequently, electrophoretic mobility-shift assay (EMSAs) of nuclear extracts from ALP-treated and untreated glEND.2 cells were prepared as described . In brief, cells were resuspended in 200 μl of hypotonic lysis buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol) and incubated on ice for 15 minutes before the addition of 5 μl of 10% Nonidet P40. After vortex-mixing, the nuclei of the lysed cells were collected by centrifugation, washed once in buffer A and subjected to a subsequent protein extraction procedure by vortex-mixing vigorously in 50 μl of hypertonic extraction buffer B (10 mM HEPES pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol). The nuclear proteins were obtained by collecting the supernatants after centrifugation. The protein concentration of each extract was measured with the micro-bicinchoninic acid (BCA) assay reagents from Pierce (Rockford, IL, USA).
NF-κB binding reactions were performed with 5 μg of nuclear proteins and a 32P-labelled double-stranded oligonucleotide containing the NF-κB consensus sequence derived from the mouse κ intronic enhancer (5' -GATCAGAGGGGACTTTCCGAG-3'). Approximately 20,000 c.p.m. of the labelled double-stranded oligonucleotide was applied to each binding reaction. After incubation for 10 minutes at room temperature the nuclear proteins were separated by electrophoresis on a 5% nondenaturing polyacrylamide gel. For specificity, control blocking experiments were run with unlabelled specific double-stranded oligonucleotides or a respective mutant variant (5' -CATCAGAGGCGACTTTCCGAGGGATG-3') at 10-fold and 100-fold molar excess of the labelled NF-κB probe. Subsequently, the electrophoresis gels were dried, exposed on a PhosphoImager plate and analysed with a FLA-3000 laser-based imaging system (Fujifilm Europe GmbH, Düsseldorf, Germany). For normalization of the nuclear translocation of NF-κB in a quantitative analysis, aliquots (5 μg) of the nuclear proteins were assessed in parallel for their binding activity to a 32P-labelled double-stranded oligonucleotide containing the specific recognition sequence of the constitutively active transcription factor Oct-1 (5' -CTGTCGAATGCAAATCACTAGAAG-3'). The numerical proportion between NF-κB-binding and Oct-1-binding activity as determined by the PhosphoImager in c.p.m. served as the parameter of NF-κB activation.
NF-κB-luciferase reporter assay
For transient transfection, 4× 104 glEND.2 cells grown in 24-well trays were transfected with a NF-κB reporter plasmid, pBIIx-luc [24, 25], using the Fugene6 transfection reagent (Roche Diagnostics, Mannheim, Germany). All DNA/Fugene6 incubations were performed at a ratio of 1.5 μg of DNA to 3 μl of Fugene6 in accordance with the manufacturer's recommended protocol, in DMEM (PAA Laboratories, Pasching, Austria). After 24 hours, transfected and control glEND.2 cells were treated for 2 hours with 8 to 20 μM ALP. The activation of the luciferase reporter gene was induced by 10 ng/ml IL-1β for 4 hours and monitored by the application of a commercial luciferase reporter assay. On cell lysis, luciferase activity was determined in accordance with the instructions of the manufacturer of the luciferase assay kit (Promega, Mannheim, Germany) with a luminometer (Luminat LB 9501; Berthold). Protein concentrations of cell lysates were determined with the bicinchoninic acid micro-assay method (Pierce, Bonn, Germany).