Neutrophils recruited by chemoattractants in vivo induce microvascular plasma protein leakage through secretion of TNF

Adherent neutrophils responding to chemoattractants release TNF in proximity of endothelial cell junctions to mediate microvascular leakage.


Br ief Definitive Repor t
In response to an inflammatory stimulus, prior ities change with respect to the maintenance of tissue homeostasis. Under basal conditions, the microvascular endothelium has a low perme ability to plasma proteins and forms a barrier between high intravascular and low extravas cular interstitial fluid protein concentrations. This provides an oncotic gradient that balances the opposing hydrostatic pressure gradient that would otherwise move water and small solutes from the blood to the tissues, with any small net outflow being compensated by lymphatic drain age. After the detection of potentially harmful substances, foreign organisms, or injured tissue cells, the immediate priority is to supply plasma proteins to the extravascular compartment. This delivers immunoglobulins, constituents of the complement and coagulation systems, antipro teases, and other acute phase reactants to the site to participate in local host defense and the initiation of tissue repair. The compromise to homeostasis results in tissue edema (an example of functio laesa). Tissue mast cells act as sentinel cells and provide one means of detecting po tentially harmful agents by means of an array of receptors, including Tolllike receptors and IgE bound to FcR1. Mast cells release histamine that induces plasma leakage by acting directly on histamine receptors located on the venular endothelium, the region of the microvascu lature specialized for permeability responses (Majno and Palade, 1961). Increased permea bility is dependent on a destabilization of the VEcadherin-catenin complex and its inter action with the actin cytoskeleton at EC junc tions (Yuan, 2002;Schulte et al., 2011). Other Microvascular plasma protein leakage is an essential component of the inflammatory response and serves an important function in local host defense and tissue repair. Mediators such as histamine and bradykinin act directly on venules to increase the permeability of endothelial cell (EC) junctions. Neutrophil chemoattractants also induce leakage, a response that is dependent on neutrophil adhesion to ECs, but the underlying mechanism has proved elusive. Through application of confocal intravital microscopy to the mouse cremaster muscle, we show that neutrophils responding to chemoattractants release TNF when in close proximity of EC junctions. In vitro, neutrophils adherent to ICAM-1 or ICAM-2 rapidly released TNF in response to LTB 4 , C5a, and KC. Further, in TNFR / mice, neutrophils accumulated normally in response to chemoattractants administered to the cremaster muscle or dorsal skin, but neutrophil-dependent plasma protein leakage was abolished. Similar results were obtained in chimeric mice deficient in leukocyte TNF. A locally injected TNF blocking antibody was also able to inhibit neutrophil-dependent plasma leakage, but had no effect on the response induced by bradykinin. The results suggest that TNF mediates neutrophil-dependent microvascular leakage. This mechanism may contribute to the effects of TNF inhibitors in inflammatory diseases and indicates possible applications in lifethreatening acute edema. chemoattractantinduced increased microvascular permea bility and describe a potential mechanism for neutrophil dependent edema.

RESULTS AND DISCUSSION Neutrophil chemoattractants induce generation of TNF in vivo
As part of our investigations into the impact of proinflam matory mediators on venular morphology in vivo (Voisin et al., 2010;Proebstl et al., 2012), we noted that locally ad ministered neutrophil chemoattractants can induce pericyte shape change in mouse cremaster postcapillary venules in a TNFdependent manner (Fig. 1, A and B). Specifically, intra scrotal administration of the chemoattractants LTB 4 , C5a, and KC (CXCL1) caused pericyte shape change in WT mice, as indicated by an increase in gaps between adjacent pericytes. These effects were totally absent in TNFR / mice ( Fig. 1 B), suggesting that chemoattractants can induce the generation of endogenous TNF in close proximity to venular walls. As expected, the response to exogenous TNF was also abolished in the TNFR / mice ( Fig. 1 B). LTB 4 , C5a, and KC did not induce pericyte shape change in neutrophildepleted mice ( Fig. 1 C), indicating that the generated TNF was derived from neutrophils.

TNF is associated with neutrophils adherent to venular ECs in LTB 4 -stimulated tissues in vivo
To gain a better understanding of the functional role of endogenously generated TNF, we sought to investigate the site and dynamics of TNF release in response to neutrophil chemotactic mediators in vivo. Initial in vitro studies pro vided direct evidence for the existence of preformed intracel lular TNF stores in mouse blood neutrophils and the ability of these cells to rapidly release TNF in response to LTB 4 (Fig. 2,A and D). This was shown by the timedependent loss of intracellular TNF in neutrophils stimulated with LTB 4 (Fig. 2, A, B, and D) and by the detection of soluble TNF in super natants of these cells (Fig. 2 C). To investigate the dynamics of TNF release from neutrophils within microvessels of chemoattractantstimulated tissues in vivo, a protocol was developed for detecting expression of TNF during early neutrophilvenular wall interactions. Cremaster muscles of Lys-EGFP-ki mice were examined by confocal intravital microscopy as previously detailed (Woodfin et al., 2011). Fur thermore, in vivo immunostaining of EC junctions using an anti-PECAM1 mAb allowed tracking of GFP high neutrophil responses within the venular lumen, neutrophil transendo thelial cell migration (TEM), and neutrophil subEC migra tion (Woodfin et al., 2011;Proebstl et al., 2012). This previously established protocol was extended to enable us to simultane ously visualize the localization of TNF as detected by an i.v. injected fluorescently labeled antiTNF mAb. Using this ap proach, no TNF was detected during the early steps of neu trophil adhesion and crawling along the luminal side of ECs after topical LTB 4 application (Fig. 2, E and F). However, TNF could be detected on the surface of neutrophils a few minutes such mediators acting directly on ECs include bradykinin, peptidoleukotrienes, plateletactivating factor, and VEGF.
Inflammatory stimuli also trigger the local production of chemoattractants that induce acute neutrophil infiltration. Once accumulated and interacting with the potentially inju rious stimulus, these cells were generally accepted to con tribute to tissue swelling in the later stages of inflammation by releasing substances that damage the endothelium, such as proteases and reactive oxygen species. Subsequent observa tions suggested another link between neutrophils and increased microvascular permeability by a mechanism that could oper ate in the very early stages of the inflammatory response. The neutrophil chemoattractants C5a, leukotriene B 4 (LTB 4 ), and fMLP were found to induce significantly increased EC per meability within 6 min of injection into animal skin, and these responses were absent in animals depleted of their cir culating neutrophils (Wedmore and Williams, 1981). This led to the conclusion that neutrophils responding to a chemo attractant signal can rapidly increase the permeability of EC junctions when the leukocytes are in close apposition to ven ular ECs; thus, regulating the supply of plasma proteins to the extravascular space. This concept was supported by studies in which a blocking anti 2 integrin mAb was found to inhibit neutrophildependent edema (Arfors et al., 1987). Collectively, neutrophil chemoattractants, including chemokines, have been found to increase microvascular permeability in several animal species (Wedmore and Williams, 1981;Williams and Jose, 1981;Björk et al., 1982;Tokita and Yamamoto, 2004), and responses to intradermal C5a have been shown to be mark edly reduced in neutropenic human subjects (Williamson et al., 1986;Yancey et al., 1987).
The perivascular basement membrane (BM) is permeable to plasma proteins and allows their entry into tissues when EC junctions open. Large colloidal molecules administered into the systemic circulation are, however, trapped under the BM/pericyte layer, which is the basis of the vascular labeling technique that first identified venules as the site of action of histamine (Majno and Palade, 1961). Some early studies showed that neutrophils can disrupt the perivascular BM so that large colloidal molecules can pass through (Hurley, 1964;Huber and Weiss, 1989). These observations may relate to our previous findings that show changes in BM morphology and enlarged gaps between pericytes induced by inflamma tory mediators in vivo (Wang et al., 2006;Voisin et al., 2009;Proebstl et al., 2012). In extending these findings, we noted that neutrophil chemoattractants induced pericyte shape change in vivo in a neutrophildependent manner, and that this response was mediated by endogenously generated TNF. Because, in response to chemoattractants, neutrophils within the vascular lumen expressed TNF in close proximity of EC junctions and TNF increases microvascular permeabil ity to plasma proteins (Brett et al., 1989), we investigated if neutrophilderived TNF could also provide an important link between neutrophils and increased endothelial perme ability. The present results provide direct evidence for the ability of endogenous neutrophilderived TNF to mediate Br ief Definitive Repor t adherent neutrophils corresponded to 43-88% of the total intracellular level of TNF. Collectively, these results demon strate that EC  2 integrin ligands can positively regulate TNF release from neutrophils although the extent of this priming effect is stimulusspecific.

Endogenous TNF mediates neutrophil-dependent increased microvascular permeability in vivo
As TNF expression was associated with neutrophilvenular wall interactions, we postulated that the release of this cytokine may change the barrier function of the EC monolayer. Locally ad ministered neutrophil chemoattractants induced neutrophil infiltration into the cremaster muscle in WT mice, which were not significantly different in TNFR / mice ( Fig. 4 A).
These results indicate that endogenous TNF does not play a key role in mediating acute neutrophil infiltration induced by neu trophil chemoattractants in this model. However, vascular leak age induced by LTB 4 in the cremaster muscle, quantified as the local accumulation of i.v. injected Evans Blue, was abolished in TNFR / mice (Fig. 4 B). This phenomenon was also investi gated in the mouse dorsal skin, where depletion of circulating neutrophils blocked microvascular leakage induced by LTB 4 , but had no effect on the response to exogenous TNF (Fig. 4 C). In the dorsal skin, LTB 4 , C5a, and KC were all found to induce rapid and transiently increased microvascular permeability, with the greatest plasma protein leakage being noted within the first 30 min after injection (Fig. 4 D). As observed using LTB 4 in the cremaster muscle, microvascular leakage induced by all of these stimuli was inhibited in the dorsal skin of TNFR / mice ( Fig. 4 E), and chimeric mice generated by bone marrow transfer of TNF / hematopoietic stem cells into WT recipients ( Fig. 4 F). The latter results specifically indicate the importance before TEM, a response that was sustained during the breach ing of the endothelium and remained elevated during neu trophil subEC crawling for observation periods of up to 45 min (Fig. 2, E and F). No such increase in signal was de tected in mice injected with a control mAb. Collectively, these results provide direct evidence for the ability of LTB 4 to stim ulate a rapid release of TNF from neutrophils in vitro and in vivo. In vivo, this response occurred when neutrophils were in close association with the venular wall, suggesting that neutrophil-EC interactions facilitate TNF release/cell surface expression and that endogenously generated TNF may regulate early neu trophildependent microvascular responses. These key issues were addressed in the studies detailed below. Neutrophils in suspension, or adherent to ICAM-1 or ICAM-2, can rapidly release TNF in response to chemoattractants Previous studies have indicated the importance of  2 integrin mediated neutrophilEC adhesion in neutrophildependent increased microvascular permeability (Arfors et al., 1987;Gautam et al., 2000). To investigate the impact of this pathway on TNF release, purified neutrophils in suspension or adher ent to ICAM1 or ICAM2 (key EC  2 integrin ligands), were stimulated with LTB 4 , C5a, or KC, and supernatants were assayed for TNF. LTB 4 was clearly able to stimulate TNF release from neutrophils in vitro and engagement of ICAM1 or ICAM2 was not a prerequisite for secretion under the conditions of these experiments, although there was a trend toward enhanced and/or accelerated TNF release from neu trophils adherent to ICAMs (Fig. 3 A). In contrast to LTB 4 , the stimuli KC and C5a induced significant levels of TNF release from neutrophils only when the cells were adherent to ICAM1 or ICAM2 (Fig. 3 B). TNF secreted from stimulated (A) Representative confocal images of postcapillary venules from five independently conducted control (PBS) or chemoattractantstimulated cremaster muscles (4 h) labeled for pericytes (SMA). Bar, 20 µm. (B) Pericyte gap size was quantified in cremasteric postcapillary venules of WT and TNFR / mice injected with PBS (n = 10 and 5, respectively), LTB 4 (n = 12 and 4, respectively), C5a (n = 5 and 4, respectively), KC (n = 5 and 4, respectively), or TNF (n = 4 and 3, respectively) for 2-4 h, involving 12 independent experiments. (C) As above but using control mice (n = 3 for PBS and LTB 4 , n = 5 for C5a, n = 6 for KC) or mice depleted of their circulating neutrophils (n = 3 mice/ group), involving 5 independent experiments. Six vessel segments per mouse were analyzed. Data are means ± SEM. Significant differences from PBS-treated tissues or other statistical comparisons (indicated by lines) are shown by asterisks. ***, P < 0.001. of neutrophilderived TNF in chemoattractantinduced mi crovascular leakage. Exogenous TNF was an effective inducer of increased microvascular permeability in the skin, a response that was undetectable in TNFR / mice, but as expected, was normal in the chimeric mice (Figs. 4,E and F). In line with our overall hypothesis that TNF mediates neutrophildependent increased microvascular permeability, exogenous TNF induced rapid plasma protein leakage. Specifically, the bulk of the re sponse occurred within the first 30 min (Fig. 4 G) and signifi cant vascular leakage was detected as early as 15 min after local administration of TNF (Fig. 4 H). A blocking antiTNF mAb, when coinjected with LTB 4 , also inhibited increased microvas cular permeability, but this protocol had no impact on vascular leakage induced by the neutrophilindependent permeability increasing mediator bradykinin (Fig. 4 I).

A model for neutrophil-dependent edema
In summary, we provide evidence for the involvement of endogenously generated TNF as a mediator of neutrophil dependent increased microvascular permeability. Several permeabilityincreasing substances have been proposed as mediators of this process, including TNF (DiStasi and Ley, 2009), but direct evidence for a conclusive mechanism has been lacking. We propose the following sequence of events within the context of an acute inflammatory response. Chemo attractants generated in tissues in response to an inflamma tory stimulus engage with their receptors on neutrophils within the venular lumen and trigger  2 integrin activation. This induces rapid neutrophil adhesion, crawling on the ven ular wall, and the initiation of TEM mediated by the  2 inte grin EC ligands ICAM1 and ICAM2 (Phillipson et al., 2006;Ley et al., 2007;Woodfin et al., 2009;Halai et al., 2014). When The associated TNF detected on the leukocyte surface for each step (intensity rainbow color code from blue [low intensity] to red [high intensity]) is shown in the bottom panels with and without the associated neutrophil. Bar, 5 µm. (F) Mean fluorescent intensity of signals on neutrophils was quantified in mice injected with anti-TNF or control mAbs (n = 4 mice/group) from 4 independent experiments. A total of 40 neutrophils were tracked and analyzed for each group. All data are means ± SEM. Statistical differences from PBS (B and C), signals from luminal crawling cells (F), or other comparisons (indicated by lines) are shown by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Br ief Definitive Repor t
of anionic TNF on the venular endothelium, with both ef fects potentially contributing to a synergism between HBP and TNF.
As the perivascular BM is regarded as freely permeable to plasma proteins, the remodeling of the BM and the con comitant increases in gaps between adjacent pericytes that we observed (the starting point in this study) may have little ef fect on the rate of passage of plasma proteins into the tissues. Such changes in pericyte and BM morphology may be more relevant to the extravasation of bloodborne cells, micro organisms, or high molecular weight immune complexes under certain circumstances.
These observations provide an explanation for the rapid neutrophildependent microvascular plasma protein leakage induced by neutrophil chemoattractants in vivo (Wedmore and Williams, 1981) and its inhibition by an anti 2 integrin mAb (Arfors et al., 1987). As in the mouse, human neutro phils are also able to secrete TNF (Smedman et al., 2009). Thus, therapeutic TNF inhibitors may owe part of their efficacy in chronic inflammatory diseases to the mechanisms postulated here. Further, the use of TNF inhibitors might be considered, or revisited, for other conditions, particularly those where acute microvascular leakage is a lifethreatening component.

MATERIALS AND METHODS
Mice. TNF receptors p55 and p75 double knockout mice (TNFR / ) on a C57BL/6 background were from The Jackson Laboratory and C57BL/6 WT mice from Harlan-Olac were used as controls. LysEGFPki mice (backcrossed to C57BL/6 for at least eight generations), exhibiting EGFP high fluorescent neutrophils (Faust et al., 2000), were used with the permission of T. Graf (Albert Einstein College of Medicine, Bronx, NY) and were pro vided by M. Sperandio (Ludwig Maximilians University, Munich, Germany). Chimeric mice deficient in leukocyte TNF were generated by lethal irradia tion of C57BL/6 WT mice (5.5 Gy twice, 4 h apart) and injection of bone marrow cells (1.5 × 10 6 cells/recipient i.v.) from TNF / mice (gift from V. Quesniaux, Centre National de la Recherche Scientifique Orleans, France). C57BL/6 WT littermates receiving WT bone marrow were used as con trols. Mice were used between 6-12 wk of age. All animal experiments were conducted according to The Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 (SI 2012/3039), all projects are reviewed and approved by the Animal Welfare and Ethical Review Board (AWERB) within Queen Mary University of London. the neutrophils are undergoing TEM or are located on the abluminal surface of ECs, chemoattractants induce the rapid surface expression and release of preformed TNF from the leukocytes, a response that can be upregulated by adhesive interactions with ECs. The binding of TNF to its receptors on ECs stimulates the phosphorylation and endocytosis of the VEcadherin complex in the junctions (Schulte et al., 2011) together with actomyosin contraction (Yuan, 2002), resulting in the opening of EC junctions and the leakage of plasma proteins. The signaling pathways that mediate TNFinduced increased EC permeability have been extensively studied and appear to involve activation of the small GTPase Rac (Cain et al., 2010) and the phosphatidylinositol (3,4,5)trisphosphatedependent Rac exchanger 1 (PRex1; Naikawadi et al., 2012).
As TNF is both secreted from neutrophils and is a neutro phil secretagogue, it is potentially possible that under some inflammatory conditions TNF may act cooperatively with other neutrophilderived EC permeability factors. Of im portance, we have found that neutrophildependent micro vascular leakage is unaffected in neutrophil elastase / mice and in animals treated with ROS inhibitors (unpublished data). However, other factors may act synergistically with TNF, e.g., neutrophilderived heparinbinding protein (HBP; CAP37, azurocidin), which has been implicated in neutrophil dependent plasma leakage (Gautam et al., 2001;Di Gennaro et al., 2009). High concentrations of recombinant HBP have been shown to increase endothelial permeability, a charge dependent effect (Gautam et al., 2001) similar to that of other very basic molecules such as polylysine (Needham et al., 1988). Supernatants from LTB 4 stimulated neutrophils have been reported to increase endothelial permeability, a response lost after incubation of supernatants with a solid phase poly clonal antibody to HBP (Di Gennaro et al., 2009). However, based on our observations, such supernatants would be ex pected to contain TNF, which may be removed by the anti body as a consequence of ionic interactions between highly positively charged HBP and the negatively charged cytokine. At lower concentrations, HBP is able to augment neutrophil adhesion (Lee et al., 2003) and may facilitate the retention Figure 3. Neutrophils in suspension or adherent to ICAM-1 and ICAM-2 release TNF in response to chemoattractants. Purified neutrophils in suspension or after adhesion to ICAM-1-or ICAM-2-coated plates were stimulated with LTB 4 for 15 (n = 5, 7, and 7 samples, respectively) or 30 min (n = 4, 6, and 6 samples, respectively; A) or KC and C5a (n = 3) for 30 min (B). Control samples (in suspension or adherent to ICAM-1 o ICAM-2) were treated with PBS (n = 6, 7, and 7, respectively). Supernatants were assayed for TNF by ELISA. Graphs show means ± SEM of 3 independent experiments. ND, not detected. Significant differences from PBS control are indicated by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001. captured using a LSM 5 PASCAL confocal laserscanning microscope (Carl Zeiss) incorporating a 40× waterdipping objective (numerical aperture 0.8, resolution 0.37 µm). Zstack images of half postcapillary venules (20-45 µm diam) were captured using the multiple track scanning mode at a resolution of 800 × 800 pixels in the x × y plane. The size (area) of gaps between adjacent pericytes and the number of transmigrated neutrophils were analyzed in postcapillary venules using ImageJ (National Institutes of Health) or IMARIS (Bitplane), respectively, as described previously (Wang et al., 2006;Voisin et al., 2010). In brief, 3Dreconstructed confocal images of half vessels (vessels were split along the longitudinal axis) were artificially displayed on a 2D surface (zprojection, maximum intensity) and converted into 8bit grayscale projec tions. Gaps between adjacent pericytes (SMAnegative regions) were either manually encircled across 200µm vessel segments, or automatically quantified by ImageJ. The values of all ROI areas/vessel segment were cumulated and an aver age unit area of at least 4 vessel segments per mouse were quantified and plotted Induction of cremasteric inflammatory reactions and ex vivo analysis of tissues by confocal microscopy. Male mice were anaesthetized by i.m. injection of 1 ml/kg ketamine (40 mg)/xylazine (2 mg) in saline. LTB 4 (30 ng; Merck), C5a (300 ng; R&D Systems), KC (500 ng; AbD Serotec), or TNF (300 ng; R&D Systems) in 300 µl PBS or PBS alone were administered intra scrotally. Reaction times were as indicated, i.e., 2 h (KC and TNF) or 4 h (LTB 4 , C5a, and IL1). At the end of each in vivo test period, mice were sacrificed and cremaster muscles were prepared for immunofluorescence staining and subsequent ex vivo analysis as previously described (Wang et al., 2006). In brief, cremaster muscles were fixed in 4% paraformaldehyde at 4°C, blocked and permeabilized in 10% normal goat serum, 10% FCS, and 0.5% Triton X100 at room temperature. To visualize pericytes and neutrophils, tissues were incubated with primary antibodies anti-SMACy3 (1A4; Sigma Aldrich) and anti-MRP14 (gift from N. Hogg, Cancer Research UK, Lon don, England) conjugated to Alexa Fluor 647. 3D confocal images were or BK when co-injected with a control mAb (n = 10) or an anti-TNF blocking mAb (n = 8) using a 30 min reaction time from 3 independent experiments. All datasets are means ± SEM. Statistically significant differences from PBS treatment or other comparisons (indicated by lines) are shown by asterisks. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
at 56°C for 24 h. The amount of accumulated Evans blue in the tissue super natant was quantified by spectroscopy at 620 nm.
Isolation and adhesion assay of blood neutrophils. Murine blood neutrophils were isolated using an anti-Ly6G MicroBead kit (MACS; Miltenyi Biotec) according to the manufacturer's protocol (93% neutrophil purity was obtained as determined by flow cytometry). Cells, pooled from 4-6 mice (2 × 10 5 cells in 100 µl RPMI containing 10% FCS and 25 mM HEPES), were seeded onto 16well LaboratoryTek glass chamber slides (Thermo Fisher Scientific) precoated with rhICAM1 (2.5 µg/ml) or rmICAM2 (10 µg/ml; both from R&D Systems) and allowed to adhere for 30 min at 37°C before being stimulated with LTB 4 (10 nM), KC (3 nM), or C5a (3 nM). Control cells received PBS only. For comparison, purified neutro phils kept in suspension in LoBind tubes (Eppendorf) were also stimulated with chemoattractants or PBS. Supernatants were taken after 15 or 30 min stimulation and assayed for TNF by ELISA (eBioscience; sensitivity of 8 pg/ml). Total neutrophil intracellular content of TNF was quantified as 25 pg/ml as measured in cell lysates of samples containing 2 × 10 6 neutrophils/ml. Ad hesion to slides was quantified by DAPI staining and IMARIS analysis.
Stimulation and analysis of blood leukocytes by flow cytometry and confocal microscopy. Whole blood was collected from WT mice and red blood cells were lysed with AKC lysis buffer (150 mM NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA). Mixed blood leukocytes, pooled from 2-3 mice (2 × 10 6 cells in 1 ml) were stimulated with 10 nM LTB 4 in PBS com plemented with 0.25% FCS, 1 mM CaCl, 1 mM MgCl, and 5 mM glucose for 10 or 30 min at 37°C. Supernatants were recovered for ELISA and stored at 80°C. Cells were washed twice at 500 g for 10 min at 4°C and analyzed by flow cytometry or confocal microscopy. For flow cytometry cells were costained for different leukocyte surface markers using the following anti bodies: anti-Ly6GPE (1A8; BioLegend; neutrophils), anti-CD115A488 (AFS98; eBioscience; monocytes), anti-CD45PE/Cy5 (RA36B2; Bio Legend; panleukocyte marker), and anti-CD3ePECy5 (1452C11; eBiosci ence; T cells). For intracellular cytokine detection and MRP14 labeling (for confocal microscopy only), cells were fixed and permeabilized using BD Cy tofix/Cytoperm Kit (BD), and stained with an antiTNF Ab (MP6XT22), or isotypematched control Ab (conjugated to Alexa Fluor 647 or 555, re spectively), or antiMRP14 Ab conjugated to Alexa Fluor 488. For confocal microscopy cells were further stained with the nuclear dye DRAQ5. Surface and intracellular expression of molecules of interest were measured by flow cytometry on a FACSFortessa (BD) and analyzed using FlowJo software (Tree Star) (neutrophils were gated as Ly6G + CD45 + CD115  CD3  cells) or a LSM 5 PASCAL confocal laserscanning microscope (Carl Zeiss) incor porating a 63× oildipping objective (NA 1.4) analyzed with IMARIS.
Statistical analyses. Data are presented as means ± SEM of at least 3 inde pendent experiments. Statistical differences between groups were analyzed using Student's t test or ANOVA followed by Dunnett or Bonferroni multiple comparison tests. P values <0.05 were considered significant. as mean per mouse. Neutrophil transmigration was quantified by counting MRP14 + cells around and within 50 µm from both sides of the vessel. At least 6 vessel segments per mouse were quantified and plotted as mean per mouse.

Neutrophil depletion.
In some experiments, mice were depleted of their circulating neutrophils before tissue stimulation as previously described . In brief, antiGR1 antibody (25 µg/day; RB68C5; BD) in 500 µl saline was injected i.p. for 3 consecutive days. Control mice received rat IgG2b isotype control antibody (AbD Serotec). The level of neutrophils and monocytes was determined by flow cytometry (Gr1 + and Gr1 + /CX 3 CR 1 GFP + cells, respectively) from blood samples taken from the tail vein before and after antibody treatment. Mice treated with antiGr1 antibody showed >85% depletion of their circulating neutrophils with no impact on the pro portion of blood Gr1 + /CX 3 CR 1 GFP + monocytes.
Intravital confocal microscopy. Visualization of TNF release by neutro phils in vivo was analyzed by intravital confocal microscopy. Male LysEGFPki mice, anaesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), received an i.v. injection of a nonblocking dose of an antiTNF mAb (4 µg, MP6XT22; eBioscience) directly conjugated to Alexa Fluor 647, or rat IgG1 isotype control antibody. Cremaster muscles were prepared for intravital imaging as previously described (Woodfin et al., 2011;Proebstl et al., 2012) and superfused with Tyrode's solution containing 10 8 M of LTB 4 for 2 h. Zstack images of postcapillary venules were captured using an SP5 confocal microscope (Leica) incorporating a 20× waterdipping objec tive (NA 1.0) for 2 h. Images were acquired every minute for a total duration of 45 min with sequential scanning of different channels at a resolution of 1,024 × 512 pixels in the x × y plane and 0.7µm steps in zdirection. Label ing of ECs was performed by intrascrotal injection of a nonblocking anti-PECAM1 mAb conjugated with Alexa Fluor 555 (C390; eBioscience; 2 µg/mouse) at least 2 h before the exteriorization of the cremaster muscle. 4D confocal image sequences were then analyzed offline using IMARIS software, enabling the dynamic interaction of neutrophils with ECs to be observed, tracked, and analyzed in 3D. Specifically, 40 isolated cells from n = 4 animals for each group (i.e., i.v. injected antiTNF or control Ab treated animals) were tracked during luminal crawling, transendothelial cell migration, and, finally, subendothelial cell (abluminal) motility within the vessel wall. An isosurface volume was generated within the leukocyte chan nel (i.e., EGFP) for every time point of the indicated steps of the transmigra tion response. Mean fluorescent intensity (MFI) of the TNF/isotype control Ab channel within the leukocyte surface was quantified over time (i.e., within 45 min after LTB 4 stimulation) and normalized to the first step of transen dothelial migration (i.e., formation of an EC junctional pore within the PECAM1 channel). For clarity, the results of TNF MFI on the leukocyte are shown grouped for four different steps of the transmigration cascade, namely luminal crawling of neutrophils along the endothelium (15 min before breaching the EC layer), pretransendothelial cell migration (preTEM) phase (6 min before breaching the endothelium), TEM phase (duration of 6 min), and abluminal/subendothelial crawling (>20 min).
Permeability assay. Vascular leakage was assessed using the Mile's assay as pre viously described (Colom et al., 2012). In brief, mice were anaesthetized by i.p. injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Evans blue dye (0.5% in PBS, 5 µl/g) was injected i.v. and LTB 4 (30 ng), C5a (300 ng), KC (500 ng), TNF (300 ng), and bradykinin (100 µg; SigmaAldrich) in 50 µl PBS or PBS alone were administered intradermally into the back skin or intrascro tally and analyzed for the indicated in vivo test periods. In some experiments, tissues were coinjected with the stimulus of interest and an antiTNF block ing antibody (100 µg, MP6XT22; eBioscience) or an isotypematched control mAb. To analyze the rate of vascular leakage over certain time windows (0-30, 30-60, 90-120, and 210-240 min of the inflammatory response), inflammatory stimuli were injected intradermally at different timepoints (4, 2, 1, and 0.5 h) before the animals were sacrificed. Evans blue was injected for the last 28 min of the experiment. At the end of the experiment, animals were sacrificed, and sites of stimulation were harvested and incubated in formamide (SigmaAldrich)