Animals
Experiments were conducted on age-matched male and female Wistar rats (250–300 g; 8–10 weeks, Charles River) or C57BL6 mice (20–25 g; 8–10 weeks, Charles River) and approved by Animals Scientific Procedures Act (UK). For intravital microscopy, rats were kept on a restricted diet for 12 h to reduce intestinal motility during imaging.
Intravital microscopy
Anaesthetized rats (pentobarbitone 60 mg/kg, ip) were prepared for intravital microscopy (IVM), as previously described [16]. Rats were placed on a heated (37 °C) viewing stage, and a loop of intestine was exposed to visualize the mesenteric microcirculation. The mesentery was superfused with Tyrode’s solution (5 % CO2, pH 7.4). Images were recorded using Pinnacle Studio software (v.9). In each animal, a single un-branched postcapillary venule (diameter 25–40 μm, length >400 μm) was selected for the study. The following parameters were measured: leukocyte flux, rolling velocity, adhesion, and emigration. Rolling leukocytes were observed as cells moving visibly slower than red blood cells and were measured by counting the number of rolling leukocytes passing a fixed reference point (flux) on the vessel segment over a 2-min period. Leukocyte rolling velocity was determined from the time required for a randomly chosen leukocyte to roll across 200 μm. Rolling velocities of six leukocytes were averaged and expressed as micrometer per second. Adherent leukocytes were identified as cells that remained stationary within the vascular lumen for a period of at least 30 s and were counted in four consecutive 100-μm vessel segments. Leukocyte emigration from the postcapillary venule into the tissue was quantified by counting the number of cells up to 50, 50–100, and 100–150 μm away from the vessel wall in parallel with 100-μm vessel segments. Four readings were taken for each vessel and quantified on one side of the vessel wall. Red blood cell centreline velocity was measured in venules using an optical Doppler velocimeter (Microcirculation Research Institute, Texas A&M University, USA). Mean red blood cell velocity was calculated from centreline velocity/1.6, and venular shear rate was determined based on the Newtonian definition (8000 × mean red blood cell velocity / venular diameter).
In some experiments, male rats were treated with Cxcl5 (3 μg/kg, ip, R&D Systems) and IVM conducted at 2 h.
Mesenteric ischemia/reperfusion
Rats from the same litter were alternately assigned to sham or I/R group, with experiments conducted on males and females on alternate days. Ischemia was induced by occluding the superior mesenteric artery (SMA). After 30 min of ischemia, reperfusion was permitted for up to 2 h and leukocyte dynamics were measured by IVM every 15 min. Sham-operated animals underwent identical surgical procedures without occlusion of the SMA. Blockade of endogenous Cxcl5 was achieved by 1-h pre-treatment with Cxcl5 monoclonal antibody (20 μg/kg, iv, R&D Systems).
Histology of mesentery
The intestinal wall was removed and portions of mesentery were fixed (4 % paraformaldehyde, 5 min) and dried onto slides overnight. Tissues were immersed in absolute alcohol (5 min) and washed with distilled water prior to staining with hematoxylin and eosin.
Determination of intestinal necrosis
Following reperfusion, a 4-cm segment of the exposed intestinal wall was purged, cut into 2–3 mm cubes and incubated with p-nitroblue tetrazolium dye (NBT, 0.5 mg/ml, 20 min, 37 °C). Necrotic unstained portions were separated from viable stained tissue and expressed as a percentage of total wet weight of the segment.
Lung myeloperoxidase activity
Lung tissues were gently rinsed, homogenized in 0.5 % (w/v) hexadacyl trimethylammomium bromide and centrifuged (13,000g, 10 min, 4 °C). Peroxidase activity of the supernatant was measured as the rate of H202-dependent oxidation of 3,3′,5,5′-tetramethylbenzidine relative to purified myeloperoxidase (MPO), by optical density (620 nm). Tissue MPO levels were normalized to total protein content.
Collection and preparation of leukocytes and mesenteric tissue
Blood leukocytes were collected into 0.5 M EDTA. Peritoneal leukocytes were collected by lavage (10 ml PBS/0.25 % BSA/2 mM EDTA). Bone marrow (BM) cells were isolated from the femur by flushing with ice-cold PBS (rats, 5 ml; mice, 1 ml) and passing through a 70-μm cell strainer. Plasma and supernatants were collected by centrifugation (300g, 5 min, 4 °C) and stored at −80 °C for protein analysis. For all cell samples, erythrocytes were lysed and leukocytes were either snap frozen for RNA analysis or prepared for flow cytometry. Mesenteric tissues were isolated by separating the mesentery from the intestinal wall, snap frozen, and stored at −80 °C for RNA analysis.
Flow cytometry
Leukocytes were resuspended in PBS/1 % goat serum (2 × 106 cells/ml). Flow cytometry was conducted on BD FACScalibur™ with data analyzed by FlowJo 7.6.1. Leukocytes were fixed and permeabilized (Leucoperm, AbD Serotec) and incubated with antibodies (30 min, 4 °C) to leukocyte subset markers, integrins, or L-selectin (in live cells), using respective isotype antibodies as controls and compensated as appropriate for multiple labeling. Surface integrin and L-selectin expression was calculated as fold expression compared to isotype (relative fluorescence intensity: RFI). For additional details about antibodies, see Additional file 1: Table S1. Neutrophil shape change was assessed by increased ability of RP1+ leukocytes to scatter light in flow cytometer and expressed as percentage of neutrophils exhibiting high forward scatter (FSChi), as previously described [17, 18].
Quantification of chemokines and cytokines
RNA was extracted from leukocyte pellets and mesenteric tissue (NucleoSpin, Macherey-Nagel), reverse transcribed (Mouse Moloney Leukaemia Virus reverse transcriptase) and 20 ng cDNA submitted to quantitative real-time PCR (Applied Biosystems 7900HT), and quantified using SYBR® green (for primer sequences, see Additional file 1: Table S2). RNA levels of target genes were assessed by threshold cycle number (Ct) and normalized to Ct of house-keeping gene for 18S and calculated as fold expression relative to the mean Ct value of the control group, using ∆∆Ct method [19]. Chemokines in plasma, cell-free BM washouts, or blister fluid were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (R&D Systems: Cxcl1, Cxcl5, CXCL6; eBioscience: Ccl2).
In vitro stimulation of leukocytes
BM leukocytes (5 × 105) in phenol red-free RPMI medium (10 % fetal calf serum, 20 mM L-glutamine, 1000 U/ml penicillin/streptomycin) were plated in 48-well plates and stimulated with Cxcl1 or Cxcl5 (10 ng/ml, 2 h, R&D Systems). Cells were collected using EDTA cell dissociation buffer (Invitrogen).
Leukocyte adhesion assay
BM leukocytes (105 cells) were prepared in serum-/phenol red-free RPMI medium and stimulated with Cxcl5 (100 ng/ml, 1 h, R&D systems) prior to plating in black opaque 96-well plates coated with BSA (2 % w/v) or ICAM1 (1 μg/well) for 40 min (37 °C). Plates were prepared by coating wells with protein overnight and treated with BSA (1 % w/v, 1 h) to inhibit non-specific interactions prior to addition of leukocytes. Non-adherent leukocytes were removed by washing with Ca2+/Mg2+-free PBS. Adherent leukocytes were labeled with calcein AM (5 μM, 1 h), quantified by spectrophotometry (absorbance 485 nm), and expressed as a percentage of absorbance of 105 labeled cells.
Carrageenan pleurisy
Pleurisy was induced in Wistar rats by injection of 0.15 ml of 1 % carrageenan (w/v) into the pleural cavity. Pleural leukocytes were collected at 3 h by lavage (1 ml PBS/0.3 % citrate (w/v). Edema was assessed by the weight of excess fluid recovered from the pleural cavity.
Renal I/R
Male and female C57BL6 mice were anesthetized with ketamine/xylazine (100 mg/kg, 10 mg/kg, ip), and renal pedicles were occluded using microvascular clamps. After 30-min bilateral ischemia, the clamps were removed and the skin was sutured. Analgesic buprenorphine (0.1 mg/kg, s.c.) was administered, and mice were allowed to recover for 24 h prior to harvesting samples.
Human skin blisters
Ethical approval was obtained from University College London Ethics Board. Blisters were elicited on the ventral aspect of the forearms of healthy volunteers (eight men, six women, aged 19–32 years) by applying 10 μl of 0.1 % Cantharone (Dormer Labs, Inc.). Volunteers did not take any medications for 2 weeks before commencement of the study and abstained from exercise, alcohol, and caffeine for at least 24 h prior to induction of blister. Blister fluid was collected at 24 h and leukocytes were prepared for cytometry, as previously described [20]. After exclusion of CD3+ lymphocytes, neutrophils were identified as CD16hi/HLA-DR− and monocytes as CD14hi/HLA-DR+.
Statistical analysis
Data are expressed as mean ± sem. Comparisons between two groups were made by two-tailed unpaired Student’s t test. For comparisons between multiple groups, a one-way ANOVA was performed followed by Bonferroni’s post-test. Comparisons between time-response curves were made using a two-way ANOVA, followed by Bonferroni’s post-test. Blister samples were analyzed by non-parametric Mann-Whitney test. Statistical analysis was performed using Prism 5.0 (GraphPad Software Inc.).