Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Sussan Nourshargh ( s.nourshargh@qmul.ac.uk ). The supply of the following reagents and mice are subject to MTA agreements with the academics indicated in parenthesis: Anti-ACKR1 mAb (Dr Ulrich H von Andrian), Lyz2-EGFP-ki mice (Dr Thomas Graf) and Acta2-RFPcherry-Tg mice (Dr David Rowe).

WT C57BL/6 mice were purchased from Charles River Laboratories, UK. The Lyz2-EGFP-ki mouse colony was kindly provided by Dr Markus Sperandio (Ludwig Maximilians University of Munich, Germany) and used with the permission of Dr Thomas Graf (Center for Genomic Regulation and ICREA, Spain). These mice contain an EGFP gene that was knocked into the lysozyme M (Lyz2) locus to generate GFPmyeloid cells (GFPneutrophils, GFPmonocytes and macrophages) and were backcrossed with C57BL/6 mice for at least 8 generations (). Acta2-RFPcherry-Tg mice were previously generated on a C57BL/6 background () and contain a transgenic insertion of the RFP variant cherry under control of the Acta2 promotor and express RFPpericytes and smooth muscle cells. The Lyz2-EGFP-ki;Acta2-RFPcherry-Tg colony was obtained by crossing the Lyz2-EGFP-ki colony with the Acta2-RFPcherry-Tg colony (). Ackr1mice () and Cxcr2mice () were backcrossed onto a C57BL/6 background for at least 11 generations. Cxcr2and WT mice exhibited similar levels of circulating neutrophils as determined 4 hr after i.s. TNF injection (1135 and 1723 neutrophils/μl blood respectively, p > 0.5, n = 7-8 mice per group). The Cxcl2mice on C57BL/6 background were generated from heterozygotes from The Jackson Laboratory and were bred at the CNIC under specific pathogen-free (SPF) conditions (). All animals were group housed in individually ventilated cages under SPF conditions at the William Harvey Research Institute, Queen Mary University of London, UK. Male mice were used for studying responses in the cremaster muscle and dorsal skin and female mice were used for peritonitis experiments and analyses of the dermal ear vasculature. All experiments were carried out using 8-12-week-old mice (age and sex matched groups) and were performed in accordance with the UK Home Office legislation.

Method Details

Inflammatory response in cremaster muscles Mice were anaesthetized with 3% isoflurane and injected i.s. with 300 ng TNF or 50 ng IL-1β (both R&D Systems), whereas control mice received 400 μl PBS (2-4 hr incubation). For the analysis of total neutrophil extravasation blocking anti-CXCL1, anti-CXCL2, anti-CXCL5 (all R&D Systems) or anti-MIF mAbs (kindly provided by Dr Christian Weber, Ludwig Maximilians University of Munich, Germany) or corresponding isotype control mAbs (30 μg/mouse, R&D Systems) were injected i.s. together with TNF. For IVM analyses mAbs were applied as described in the corresponding section below.

Whole-mount IF staining Cremaster muscles or ears were fixed in 4% paraformaldehyde (PFA, Sigma-Aldrich) for 1 hr at 4°C and permeabilized and blocked in PBS containing 0.5% Triton X-100 (Sigma-Aldrich) and 25% fetal calf serum (FCS, Thermo Fisher Scientific) for 4 hr at room temperature. Subsequently, the tissues were incubated with unlabeled or fluorescently labeled primary antibodies in PBS containing 10% FCS over night at 4°C. Where required, samples were incubated with fluorescently labeled secondary antibodies in PBS containing 10% FCS for 3 hr at room temperature. Antibody conjugation to Alexa Fluor 488, 555, 647 or DyLight 405 fluorophores was carried out using labeling kits (Thermo Fisher Scientific) according to the manufacturer’s recommendations.

Confocal microscopy and image analysis −/− and GFP-Cxcl2wt/wt neutrophils in mixed chimeric mice were identified based on their MRP14+/GFP- and MRP14+/GFP+ expression profile, respectively, and normalized for neutrophil numbers in the blood. Adherent neutrophils in Cxcl2−/− chimeras were determined by IF staining and defined as luminal neutrophils attached to ECs. Endogenous chemokine expression or rmCXCL1 or rmCXCL2 binding within venular ECs and pericytes was analyzed using polyclonal anti-CXCL1 or anti-CXCL2 antibodies (R&D Systems) as described ( Woodfin et al., 2011 Woodfin A.

Voisin M.B.

Beyrau M.

Colom B.

Caille D.

Diapouli F.M.

Nash G.B.

Chavakis T.

Albelda S.M.

Rainger G.E.

et al. The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo. high junctional and CD31dim non-junctional regions) or α-SMA, respectively, and chemokine MFI values per unit area within these isosurfaces were determined. The pericyte marker expression profile was established using anti-α-SMA, anti-PDGFR-β (R&D Systems), and anti-NG2 (Millipore) antibodies. ACKR1 expression was determined using mAbs specific for ACKR1 ( Thiriot et al., 2017 Thiriot A.

Perdomo C.

Cheng G.

Novitzky-Basso I.

McArdle S.

Kishimoto J.K.

Barreiro O.

Mazo I.

Triboulet R.

Ley K.

et al. Differential DARC/ACKR1 expression distinguishes venular from non-venular endothelial cells in murine tissues. high or VE-Cadherin+ regions) or non-junctional regions (isosurface on CD31dim/VE-Cadherin- regions). All protein expression levels were quantified from 4-10 images per mouse and expressed as MFI values per unit area of tissues stained with specific antibodies subtracted by MFI values per unit area of tissues stained with appropriate isotype controls. All images used for protein and mRNA (FISH) quantifications were captured with the 63x objective in the multiple track scanning mode every 0.43 μm at a resolution of 1024 × 512 μm corresponding to a voxel size of 0.099 × 0.099 × 0.43 μm in x × y × z, respectively. The fluorescence intensity line profiles were determined using ImageJ software (NIH). Immunostained whole-mount cremaster muscles or ears were imaged with an up-right Leica TCS SP5 (Leica) or inverted Zeiss 800 (Carl Zeiss) confocal laser scanning microscope equipped with argon and helium lasers (488, 561 and 633 nm excitation wavelengths) or solid-state laser diodes (405, 488, 561 and 640 nm excitation wavelengths), respectively. Serial z stacks of postcapillary venules (diameter 20-45 μm) were acquired with a water dipping 20x (1 NA) objective or oil immersion 40x (1.3 NA) or 63x (1.4 NA) objectives and the resulting images of half vessels were reconstructed in 3D and analyzed using Imaris software (Bitplane). Neutrophil extravasation in cremaster muscles was determined by immunostaining using mAbs against MRP14 (kindly provided by Dr Nancy Hogg, Cancer Research UK, UK), CD31 (Thermo Fisher Scientific) and α-SMA (Sigma-Aldrich) to label neutrophils, ECs and pericytes, respectively. Extravasated neutrophils per field of view (330 × 160 × 45 μm) were quantified from 8-10 images per mouse. Cxcl2and GFP-Cxcl2neutrophils in mixed chimeric mice were identified based on their MRP14/GFPand MRP14/GFPexpression profile, respectively, and normalized for neutrophil numbers in the blood. Adherent neutrophils in Cxcl2chimeras were determined by IF staining and defined as luminal neutrophils attached to ECs. Endogenous chemokine expression or rmCXCL1 or rmCXCL2 binding within venular ECs and pericytes was analyzed using polyclonal anti-CXCL1 or anti-CXCL2 antibodies (R&D Systems) as described (). Briefly, EC and pericyte isosurfaces were created based on regions immunostained for CD31 (including CD31junctional and CD31non-junctional regions) or α-SMA, respectively, and chemokine MFI values per unit area within these isosurfaces were determined. The pericyte marker expression profile was established using anti-α-SMA, anti-PDGFR-β (R&D Systems), and anti-NG2 (Millipore) antibodies. ACKR1 expression was determined using mAbs specific for ACKR1 (), VE-Cadherin (Thermo Fisher Scientific) and CD31. Where indicated endothelial ACKR1 and chemokine MFI values were quantified within EC-junctions (isosurface created on CD31or VE-Cadherinregions) or non-junctional regions (isosurface on CD31/VE-Cadherinregions). All protein expression levels were quantified from 4-10 images per mouse and expressed as MFI values per unit area of tissues stained with specific antibodies subtracted by MFI values per unit area of tissues stained with appropriate isotype controls. All images used for protein and mRNA (FISH) quantifications were captured with the 63x objective in the multiple track scanning mode every 0.43 μm at a resolution of 1024 × 512 μm corresponding to a voxel size of 0.099 × 0.099 × 0.43 μm in x × y × z, respectively. The fluorescence intensity line profiles were determined using ImageJ software (NIH).

Peritoneal inflammation Peritonitis was induced by intraperitoneal (i.p.) injections of 300 ng TNF in WT mice or Cxcl2−/−, Ackr1−/− or corresponding control chimeras, whereas non-inflamed control mice received 1 mL of PBS. Blocking anti-CXCL1, anti-CXCL2, anti-CXCR2 mAbs (R&D Systems) or corresponding isotype control mAbs (3 mg/kg, R&D Systems) were administered i.v. 10 min prior to TNF. 4 hr after TNF or PBS administration, mice were culled and subjected to peritoneal lavages using 5 mL PBS containing 5 mM EDTA (Sigma-Aldrich) and 0.25% BSA. The peritoneal exudates were stained for the leukocyte marker CD45, the neutrophil marker Ly6G (both Biolegend) and the monocyte and macrophage marker CD115 (Thermo Fisher Scientific) and total numbers of infiltrating neutrophils and monocytes per peritoneal cavity were determined by flow cytometry.

Flow cytometry Where required, samples were treated with ACK buffer (150 mM NH 3 Cl, 1 mM KHCO 3 and 1 mM EDTA) to lyse red blood cells. Subsequently, the samples were incubated with anti-CD16/-CD32 antibodies (Becton Dickinson) to block Fc-receptors and stained with primary fluorescently labeled antibodies of interest. CXCR2 surface levels on neutrophils were determined with an anti-CXCR2 mAb (Biolegend). The samples were analyzed on an LSR Fortessa flow cytometer (Becton Dickinson) and FlowJo software (TreeStar). Total neutrophil numbers in samples were determined using fluorescent counting beads (Thermo Fisher Scientific).

Confocal intravital microscopy (IVM) Proebstl et al., 2012 Proebstl D.

Voisin M.B.

Woodfin A.

Whiteford J.

D’Acquisto F.

Jones G.E.

Rowe D.

Nourshargh S. Pericytes support neutrophil subendothelial cell crawling and breaching of venular walls in vivo. Woodfin et al., 2011 Woodfin A.

Voisin M.B.

Beyrau M.

Colom B.

Caille D.

Diapouli F.M.

Nash G.B.

Chavakis T.

Albelda S.M.

Rainger G.E.

et al. The junctional adhesion molecule JAM-C regulates polarized transendothelial migration of neutrophils in vivo. + neutrophils and RFP+ pericytes), were injected i.s. with an Alexa Fluor 647-labeled non-blocking anti-CD31 mAb (4 μg, clone 390, Thermo Fisher Scientific) to stain EC junctions (2 h). Alternatively, IVM was performed on bone marrow chimeric mice exhibiting Lyz2-EGFP-ki hematopoietic (GFP+ neutrophils) and Ackr1−/− or WT non-hematopoietic cells, which received i.s. injections of an Alexa Fluor 555-anti-CD31 mAb (clone 390). The mode and dynamics of neutrophil migration through blood vessel walls was analyzed by confocal IVM, as described previously (). In order to analyze neutrophil interactions with ECs and pericytes simultaneously Lyz2-EGFP-ki;Acta2-RFPcherry-Tg mice (exhibiting GFPneutrophils and RFPpericytes), were injected i.s. with an Alexa Fluor 647-labeled non-blocking anti-CD31 mAb (4 μg, clone 390, Thermo Fisher Scientific) to stain EC junctions (2 h). Alternatively, IVM was performed on bone marrow chimeric mice exhibiting Lyz2-EGFP-ki hematopoietic (GFPneutrophils) and Ackr1or WT non-hematopoietic cells, which received i.s. injections of an Alexa Fluor 555-anti-CD31 mAb (clone 390). 2 hr after i.s. administration of 300 ng TNF, 50 ng IL-1β or 400 μl PBS the mice were anaesthetized by i.p. administration of ketamine (100 mg/kg) and xylazine (10 mg/kg) and the cremaster muscles were exteriorized and pinned out flat over the optical window of a heated custom-built microscope stage. The animals were maintained at 37°C and the cremaster muscles were perfused with 37°C warm Tyrode’s solution (Sigma-Aldrich) during the experiment. Blocking anti-CXCL1, anti-CXCL2 or isotype control mAbs (3 mg/kg) were injected i.v. 10 min before i.s. TNF administration for the analysis of intraluminal and TEM neutrophil responses as indicated. For the analysis of TEM and post-TEM neutrophil responses the mAbs (30 μg) were injected i.s. 100 min after TNF administration. Postcapillary venules with a diameter of 20-45 μm were recorded for 0.5-2 hr using an upright Leica SP5 confocal laser scanning microscope and a 20x water-dipping objective (NA 1.0). Serial z stack images were acquired every 30 or 60 s and assembled offline into 3D videos using Imaris software. The dimension of the recorded area was typically 300 × 130 × 35 μm and the resulting voxel size was 0.29 × 0.29 × 0.69 μm in x × y × z. Models of half vessel were generated to clearly visualize individual neutrophils migrating through the different compartments of venular walls. Neutrophil migration mode and dynamics (speed and displacement) were determined by manual tracking of individual neutrophils using Imaris software and migratory paths were graphically illustrated using the Chemotaxis and migration tool (IBIDI). Luminal neutrophils were defined as adherent when they remained stationary on the endothelium for at least 30 s and as intraluminally crawling when they exhibited a displacement of at least 2 cell diameters on the endothelium over the entire observation period. The numbers of adherent and crawling neutrophils were expressed as means determined at 4 time points per mouse. Neutrophil TEM was defined as an event where neutrophils fully migrated through EC junctions in a luminal-to-abluminal direction. Aborted TEM was classified as a response where luminal neutrophils extended protrusions through EC junctions (but did not exhibit complete TEM) and subsequently reverse migrated in an abluminal-to-luminal direction and finally disengaged from the EC junction and re-entered the vascular lumen. Reverse neutrophil TEM was a response where sub-EC neutrophils (after having fully breached the endothelium) migrated through an EC junction in an abluminal-to-luminal direction. TEM, aborted TEM and reverse TEM events were quantified over the entire IVM observation period of 2 h.

Neutrophil isolation Murine neutrophils were isolated from bone marrow cells (harvested from tibiae and femora) or peripheral blood by negative magnetic cell sorting using the Neutrophil isolation kit (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of isolated neutrophils (CD45+/Ly6G+/CD115- expression profile) was consistently > 95% as determined by flow cytometry.

Ca2+ flux assay Up to 10 × 106 isolated bone marrow neutrophils from WT C57BL/6 mice were suspended in 1 mL RPMI-1640 medium (Sigma-Aldrich) supplemented with 5% FCS, 2 mM L-glutamine, 1 g/L NaHCO 3 , and 20 mM HEPES and incubated with 4 μM of the fluorescent Ca2+ indicator Fluo-4, 0.04% Pluronic F-127 and 1 mM Probenecid (all Thermo Fisher Scientific) for 45 min at 37°C. The neutrophils were then washed and analyzed by flow cytometry at room temperature. Specifically, Fluo-4 fluorescence readings (excitation wavelength: 488 nm, 530/30 nm bandpass filter) were recorded for 30 s to establish a baseline and for 3 min after the addition of CXCL1 or CXCL2 (Preprotech). Results show baseline corrected Fluo-4 MFI or peak fluorescence values as determined using FlowJo software.

In vitro neutrophil adhesion assay 96 well plates were coated with 2.5 μg/mL ICAM-1 (R&D Systems) over night at 4°C and blocked with 10% low endotoxin BSA (Sigma-Aldrich) for 2 hr at room temperature. Isolated bone marrow neutrophils were added, and the plates were centrifuged at 20 g for 2 min and treated with CXCL1, CXCL2 or control medium for 15 min at 37°C. Non-adherent cells were then washed away with PBS containing 1 mM CaCl 2 , 0.5 mM MgCl 2. Subsequently, adherent neutrophils were detached by TrypLE express cell detachment solution (Thermo Fisher Scientific) and quantified by flow cytometry. The results were expressed as the percentage of adherent neutrophils after chemokine addition, subtracted by the percentage of adherent neutrophils in the absence of chemokines.

Western Blot Isolated neutrophils were lysed in 1x Laemmli Buffer, denatured at 95°C and subjected to standard Western Blot analysis using anti-pan-AKT and anti-phospho-AKT primary antibodies (Cell Signaling Technology) and a horseradish peroxidase-conjugated secondary antibody (Dako). Proteins were visualized by enhanced chemiluminescence acquired on X-ray film (Fuji Medical) and quantified by ImageJ software.

Transwell chemotaxis assay Bone marrow neutrophils from Lyz2-EGFP-ki mice or mixed Cxcl2−/−-Cxcl2wt/wt chimeric mice were seeded into top chambers of Transwell plates (3 μm pore diameter, Sigma-Aldrich) in PBS supplemented with 1 mM CaCl 2 , 0.5 mM MgCl 2 , 10 mM glucose, 10 mM HEPES (Sigma Aldrich) and 0.25% low endotoxin BSA. In some experiments, Transwell filters were coated with 0.5 μg/mL CXCL1 or CXCL2 in PBS or with PBS alone over night at 4°C and blocked with 10% low endotoxin BSA for 1 hr at room temperature before adding neutrophils. Where indicated 10 nM CXCL1 or CXCL2 were added to the top chamber with the neutrophils. 0-10 nM CXCL1, CXCL2 or leukotriene B 4 (LTB 4 , Cayman Chemical) were added to the bottom chambers and the Transwell plates were incubated for 1 hr at 37°C. Neutrophils migrated into the bottom chambers were resuspended in PBS containing 5 mM EDTA and their absolute numbers were determined by flow cytometry. GFP-Cxcl2wt/wt and Cxcl2−/− neutrophils were identified by their Ly6G+/GFP+ or Ly6G+/GFP- expression profile, respectively. Confocal microscopy was used to assess chemokine immobilization on Transwell filters by immunostaining and to analyze neutrophil morphology.

Fluorescence in situ hybridization (FISH) Cremaster muscles were frozen and cut into 30 μm sections. In situ hybridization was carried out using the RNAscope fluorescent multiplex assay (Advanced Cell Diagnostics) according to the manufacturer’s instruction with slight modifications. Briefly, after dehydration the sections were incubated with Pretreat 4 for 20 min at room temperature and hybridized with probes for Pecam1, Acta2, Cxcl1 and Cxcl2 mRNAs for 2 hr at 40°C. To evaluate the assay, sections were hybridized with probes for Ppib (positive control) and DapB (negative control). The amplification steps were performed according to the RNAscope protocol. Where required, in situ hybridization procedure was directly followed by IF staining with an anti-α-SMA antibody over night at 4°C. Fluorescent mRNA spots and IF stainings were visualized by confocal microscopy. Cxcl1 and Cxcl2 mRNA copy numbers in microvascular walls were determined by automatic quantification of fluorescent spots within Pecam1- and Acta2-positive regions using Imaris software. Within this assay, as detailed in the manufacturer’s guidelines, each mRNA molecule hybridized to a probe appears as single fluorescent spot. At least 6 vessel segments per mouse were analyzed.

Pericyte isolation and culture Cremaster muscles from Acta2-RFPcherry-Tg mice were digested with 500 U/mL Collagenase II (Worthington) in PBS for 45 min at 37°C and 50 U/mL DNase I (Sigma-Aldrich) was added during the last 20 min. The resulting cell suspension was seeded onto tissue culture plates coated with gelatin and collagen I (Advanced BioMatrix) and cultured in low glucose Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FCS, 100 U/mL penicillin, 100 mg/mL streptomycin (all Thermo Fisher Scientific) and 100 pM pigment epithelium-derived factor (PEDF, Sigma-Aldrich). After ∼21 days of culture, confluent cells were detached with 5 mM EDTA and cells exhibiting the unique venular pericyte signature (α-SMA+PDGFR-β+NG2-) were isolated using the FACSCalibur cell sorter (Becton Dickinson). α-SMA+ cells were identified by RFP expression. Pericytes showing > 90% purity were subjected to further analyses.

Real-time PCR Total RNA was purified from isolated murine peripheral blood neutrophils or cultured lung ECs or cremaster muscle pericytes using the RNeasy micro kit (Quiagen) and reverse transcribed into cDNA with the iScript cDNA synthesis kit (Biorad). Quantitative real-time PCR was carried out using the iQ SYBR Green supermix (Biorad) according to the manufacturer’s protocol, primers for Cxcl1, Cxcl2 and Gapdh (Integrated DNA Technologies) and the 7900HT real-time PCR machine (Applied Biosystems). Cxcl1 and Cxcl2 mRNA levels were expressed in relation to Gapdh.

In vitro pericyte and neutrophil analysis Cultured murine primary cremaster muscle pericytes were seeded onto gelatin- and collagen I-coated plates and activated with TNF in DMEM low glucose medium containing 10% FCS and 100 U/mL penicillin and 100 mg/mL streptomycin for 4 hr at 37°C. Alternatively, isolated bone marrow neutrophils were stimulated with 1 nM TNF in PBS containing 1 mM CaCl 2 , 0.5 mM MgCl 2 , 10 mM glucose, 10 mM HEPES and 0.25% low endotoxin BSA for 1 hr at 37°C. For some experiments, neutrophils were seeded on wells that were coated with 2.5 μg/mL CXCL1 or PBS over night at 4°C and blocked with 10% endotoxin low BSA for 1 hr at room temperature. Supernatants of pericyte and neutrophil cultures were taken at the end of the stimulation periods and the cells were lysed with 1% Triton X-100 in PBS containing HALT protease and phosphatase inhibitor (Thermo Fisher Scientific). Chemokine levels were determined by ELISA kits (R&D Systems, sensitivity: 2 pg/mL for CXCL1 and 1.5 pg/mL for CXCL2).

Generation of bone marrow chimeric mice Mice exhibiting CXCL2-deficiency in the hematopoietic compartment and WT control chimeras were generated by transferring bone marrow cells from Cxcl2−/− or WT mice into Lyz2-EGFP-ki mice. Mixed Cxcl2−/−-Cxcl2wt/wt chimeras were established by transferring a 1:1 mixture of bone marrow cells from Cxcl2−/− and Lyz2-EGFP-ki mice into Lyz2-EGFP-ki mice. GFP-Cxcl2wt/wt neutrophils in mixed chimeras were distinguished from Cxcl2−/− neutrophils based on their GFP expression. Mice exhibiting GFP+ myeloid cells and Ackr1−/− or WT non-hematopoietic cells were generated by transferring bone marrow cells from Lyz2-EGFP-ki mice into Ackr1−/− or WT recipients. To generate the chimeras, recipient mice were lethally irradiated with 2 doses of 5 Gy given 4 hr apart. The following day 1.5 × 106 bone marrow cells from donor mice were injected i.v. into the irradiated mice. The chimeras were subjected to IVM analyses 4 weeks after bone marrow transplantation. Control experiments confirmed that Cxcl2−/− chimeras and mixed Cxcl2−/−-Cxcl2wt/wt chimeras showed normal circulating neutrophil numbers compared to WT control chimeras (2442, 2509 and 2410 neutrophils/μl blood, respectively, p > 0.5, n = 11-20 mice per group). Ackr1−/− and WT chimeras expressed similar levels of CXCR2 on circulating neutrophils (MFI of 1100 and 975, respectively, p > 0.5, n = 3 mice per group), as determined 3 hr after i.s. injection of TNF.

Dorsal skin inflammation TNF, LPS (Sigma-Aldrich, both 300 ng in 50 μl volumes) or PBS were injected intradermally into the dorsal skin of Cxcl2−/−, Ackr1−/− or corresponding WT control chimeric mice. After 4 h, skin samples were dissected, frozen in liquid nitrogen and homogenized in homogenization buffer (600 mM NaCl, 0.5% hexadecyltrimethylammonium bromide buffer, 600 mM KH 2 PO 4 , 66 mM Na 2 HPO 4 ) using a Precellys instrument (Bertin Technologies). Tissue debris was removed by centrifugation. The peroxidase activity in the supernatants was determined by adding the MPO substrate 3,3′,5,5′-tetramethylbenzidine (Invitrogen) and measuring the absorbance at 650 nm over 20 min using a Spectra MR photometer (Dynex technologies). The MPO activity (used as a readout for neutrophil infiltration) was expressed as the increase in optical density per min multiplied by 100.