Animal Model
Wildtype C57Bl/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany). SAP was induced by partial duct ligation and an additional caerulein injection at day 2 (50mg/kg bodyweight), as described previously6,8,12,13. To deplete Monocytes and Neutrophils mice were treated with anti-Ly6G antibody17 (BioXcell, Lebanon USA, BP0075-1), with rat anti-mouse-CCR2 antibody MC-2118 (a kind gift from Matthias Mack, Regensburg Germany, lot 1480/02) and as control with isotype IgG2a (BioXcell, BP0089) or isotype IgG2b (BioXcell BE0090). Anti-Ly6G antibody and isotype IgG2a antibody were injected i.p. with 200µg in 200µl PBS one day before and the day after duct ligation. Anti-CCR2 antibody and Isotype IgG2b antibody were injected i.p. with 20µg in 200µl PBS at the day of duct ligation and one and two days after surgery. Mice were sacrificed 72 hours after duct ligation and organs were used for analysis. Serum was stored at -80°C. Pancreas, lung and kidney were fixed in 4.5% formaldehyde and embedded in paraffin, embedded in TissueTec or frozen in liquid nitrogen for rtPCR analysis. Spleen and lung were taken for FACS analysis.
Antibodies and Reagents
The following antibodies were used for FACS staining, IHC staining and immunofluorescence staining: Anti-mouse CD4 BV650 (BioLegend 100546), anti-mouse-CD11b PerCP Cy55 (BioLegend, 101228), anti-mouse-Ly6G BV421 (BioLegend, 127628), anti-mouse-Ly6C BV605 (BioLegend 128036), anti-CD25-PECy7 (BioLegend, 102016), anti-CD69 BV510 (BioLegend, 104532), anti-CD8a BV605 (BioLegend, 100743), anti-FoxP3 APC (Miltenyi Biotec, 130-111-601), anti-mouse-CD11b (abcam, Ab133357), anti-mouse-CD68 (antibody-online, ABIN181836), anti-CCR2 (abcam, ab273050) anti-Ki67 (Bethyl, IHC-00375), anti-mouse-CD11b (abcam, Ab133357), anti-mouse-Ly6g (abcam, Ab25377), anti-mouse-Cystatin C (Novus biologicals, NB100-1033), anti-VE-cadherin (abcam, ab7047-50) anti-rabbit-HRP (DAKO, K4003), anti-mouse-HRP (DAKO, K4001). Caerulein was obtained from Sigma Aldricht (C9026-1MG, Munich, Germany), human myeloperoxidase from Calbiochem (Cat# 475911).
Flow cytometry analysis of spleen
To analyse splenocytes, spleen was taken and mashed through a 70µm cell strainer. Cells were washed with PBS and centrifuged at 300g for 6 minutes. To lyse erythrocytes pellet was resuspended in 1 ml lysis buffer for 5 minutes. After washing with PBS and centrifugation at 300g for 6 minutes cells were stained with fluorescence antibodies. Extracellular staining was performed with surface antibodies (1:50): Anti-CD4, anti-CD11b, anti-Ly6G, anti-Ly6C, anti-CD25, anti-CD69, anti-CD8a. To block non-specific Fc mediated interactions cells were pre-treated with anti-mouse CD16/CD32 antibody for 5 min (1:50). Extracellular antibodies were incubated for 30 minutes at 4°C. Cells were washed with PBS and centrifuged at 300g for 6 minutes. Pellet was resuspended in 150µl FACS buffer and measured immediately. Data were analysed with BD FACS DIVA Software and FlowJo.
Flow cytometry analysis of lung
To analyse leucocytes from lung, lungs were removed from mice and were dissociated according to the protocol of lung dissociation kit from Miltenyi (130-095-927, Bergisch Gladbach, germany). Staining for flow cytometry was performed as described in splenocyte staining. Data were analysed with BD FACS DIVA Software and FlowJo.
Serum Amylase and Lipase
Serum amylase and lipase measurement was performed with colorimetric assay from Roche/Hitacho (Amyl Ref 11876473316; Lip Ref 11821792216).
Oxygen saturation of blood
Arterial Blood was taken with a capillary from arteria carotis communis and was measured with BGA analysis machine (ABL90 FLEX, Radiometer GmbH, Germany). Carotis communis was prepared as described previously. Parameters like pO2, pCO2, glucose, lactat, etc. were measured and analysed.
MPO activity
MPO measurement was performed like previously described6,17,48,49. Lung tissue was homogenized on ice in 20mM potassium phosphate buffer (pH 7.4) and centrifuged at 14.000 rcf. The pellet was resuspended in potassium phosphate buffer (pH 6.0) containing 0.5% cetyltrimethylammoniumbromide. Suspension was frozen and thawed in 4 cycles and centrifuged at 14.000 rcf. MPO activity was measured in 50 mM potassium phosphate buffer (pH 6.0) containing 0.53 mM O-dianisidine and 0.15 mM H2O2. The MPO activity was measured as kinetic over time with a Spectramax Spectrophotometer (Molecular devices). Purified human myeloperoxidase was used as standard, the final MPO activity was calculated against protein content of the samples.
Histology, Immunohistochemistry and Immunfluorescence staining
For H&E staining the lung, pancreas and kidney were embedded in paraffin, 3µm thick slides were cut by microtome. Slides were deparaffined in Xylol, and hydrated in methanol followed by decreased ethanol concentrations, hematoxylin and eosin staining was performed as described previously13,17. For lung analysis slides were scanned with slide scanner (Pannoramic MDI BF/FL; Sysmex, Norderstedt, Germany) and analysis were made with Software Pattern Quant from Sysmex Quant Center.
For immunohistochemistry staining slides were dewaxed and cooked in antigenretrieval solution (Dako target retrieval solution, ref. S1699). Rabbit anti-mouse-CD11b antibody (abcam, cambride UK, Ab133357) was used in a concentration of 1:400 over night at 4°C. Anti-rabbit antibody was incubated for 1 hour at RT. DAB Kit (Vector, Burlingame USA, SK4100) was used to visualize CD11b positive cells.
For immunofluorescence staining TissueTec embedded slides were used. Rabbit-anti-mouse-CD11b, Ly6g, CD68, CCR2 or VE-cadherin antibody was used as 1:400. Secondary antibody was used in a concentration of 1:200. DAPI was used for nucleus staining. All slides were scanned with Pannoramic slide scanner and analysis were made with Sysmex Quant Center Nucleus or Pattern.
Cytokines in Serum
The chemokines MCP-1, MIG, IP-10, IL8 and complement system anaphylatoxins C3a, C4a and C5a were measured in human serum from patients with severe, moderate and mild form of acute pancreatitis. Measurements were performed with CBA Kits from BD (Human Anaphylatoxin Kit 561418BD; Human Chemokine Kit 552990; BD San Jose, USA). Murine chemokines and cytokines were measured by BD cytometric bead array (CBA) mouse inflammation kit in serum of mice.
Real Time Fluorescence and Deformability Cytometry
Mechanical characterization of monocytes was performed in whole blood and lung monocytes isolated from pancreatic duct-ligated mice as well as untreated controls, respectively. For monocyte identification in whole blood, cells were labeled with Ly6g (APC conjugated) and CD11b (FITC conjugated) fluorescent antibodies. Lung monocytes were isolated (lung dissociation Kit, Miltenyi) by usage of monocytes isolation kit (EasySep mouse Monocyte Isolation Kit, Stemcell, 19861A). After isolation lung monocytes are resuspended in PBS (without Ca2+ and Mg2+) complemented with 0.6% (w/v) Methylcellulose to a concentration of approximately 10 x 106 cells per ml.
High-throughput mechanical analysis was done by real-time fluorescence and deformability cytometry using the AcCellerator system with fluorescence extension (Zellmechanik Dresden GmbH)22,24. Briefly, the system consists of an inverted microscope, where a microfluidic chip with a 300 µm long constriction is assembled on a xy-stage. We use a channel cross-section of 20 µm x 20 µm for whole blood measurements and a 15 µm x 15 µm cross-section for lung monocytes. In all measurements a flow rate of 0.06 µl/s is applied. For each condition data from several thousand monocytes has been obtained. Only cells are analysed with an area ratio < 1.05 (ratio between actual cell area and cell area detected image analysis) to ensure a full representation of cell shape.
Derivation of elastic modulus was done using an analytical model published earlier23. Here, the flow profile around a moving cell is calculated assuming Stokes flow and deformation is predicted from coupling the resulting stress-distribution to the cell surface applying linear elasticity theory and assuming the cell being a homogeneous material.
Data were analysed with the Software ShapeOut.
qrt-PCR
RNA preparation was performed with Trizol as described before12. qrt-PCR was performed for NGal (5´-CACCACGGACTACAACCAGTTCGC-3´; 5´-TCAGTTGTCAATGCATTGGTCGGTG-3´). As house-keeping gene 5S (5´-GCCCGATCTCGTCTGATCTC-3´; 5´-GCCTATCAGCACCCGGTATTC-3´) was analysed and markers were normalized to 5S.
Statistical analysis
The statistical evaluation as well as the graphical presentation of the experiments were carried out in GraphPad Prism and SigmaPlot. All graphs were generated with the standard error of the mean (SE). As significance test the two-sided students T-test for unpaired samples was used, all samples p > 0.05 were classified as significant. Patients samples were analysed by Kruskal-Wallis test followed by Dunn’s multiple comparison test, significant differences were defined by p < 0.05, the correlation was tested by spearman rank order correlation. A statistical significant differences for more than 3 groups were tested by one way ANOVA followed by Tukey’s multiple comparison test, or by Kruskal-Wallis test followed by Dunn’s multiple comparison test. Statistical analysis of RT-FDC data has been performed using linear mixed models, where we compared two groups of experimental replicates, respectively50. While fixed effects account for the actual effect, i.e., increase or decrease in deformation, cell size or elastic modulus, random effects represent systematic or random measurement bias.