PMN Transendothelial Migration Research Articles

Macrophage-endothelial cell crosstalk orchestrates neutrophil recruitment in inflamed mucosa.

Neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires transendothelial migration (TEM). TEM involves several well-studied sequential adhesive interactions with vascular endothelial cells (ECs); however, what initiates or terminates this process is not well-understood. Here we describe what we believe to be a new mechanism where vessel associated macrophages (VAMs) through localized interactions primed EC responses to form ICAM-1 "hot spots", to support PMN TEM. Using real-time intravital microscopy (IVM) on lipopolysaccharide (LPS)-inflamed intestines in CX3CR1-EGFP macrophage-reporter mice, complemented by whole-mount tissue imaging and flow cytometry, we found that macrophage vessel association is critical for the initiation of PMN-EC adhesive interactions, PMN TEM and subsequent accumulation in the intestinal mucosa. Anti-colony stimulating factor 1 receptor (CSF1R) antibody-mediated macrophage depletion in the lamina propria and at the vessel wall resulted in elimination of ICAM-1 hot spots impeding PMN-EC interactions and TEM. Mechanistically, the use of human clinical specimens, TNFα knockout macrophage chimeras, TNFα/TNF receptor (TNFR) neutralization and multi-cellular macrophage-EC-PMN cocultures revealed that macrophage-derived TNFα and EC TNFR2 axis mediated this regulatory mechanism and was required for PMN TEM. As such, our findings identified clinically relevant mechanism by which macrophages regulate PMN trafficking in inflamed mucosa.

Platelet Endothelial Cell Adhesion Molecule (PECAM/CD31) Blockade Modulates Neutrophil Recruitment Patterns and Reduces Infarct Size in Experimental Ischemic Stroke

The infiltration of polymorphonuclear leukocytes (PMNs) in ischemia-reperfusion injury (I/RI) has been implicated as a critical component of inflammatory damage following ischemic stroke. However, successful blockade of PMN transendothelial migration (TEM) in preclinical studies has not translated to meaningful clinical outcomes. To investigate this further, leukocyte infiltration patterns were quantified, and these patterns were modulated by blocking platelet endothelial cell adhesion molecule-1 (PECAM), a key regulator of TEM. LysM-eGFP mice and microscopy were used to visualize all myeloid leukocyte recruitment following ischemia/reperfusion. Visual examination showed heterogeneous leukocyte distribution across the infarct at both 24 and 72 hours after I/RI. A semiautomated process was designed to precisely map PMN position across brain sections. Treatment with PECAM function-blocking antibodies did not significantly affect total leukocyte recruitment but did alter their distribution, with more observed at the cortex at both early and later time points (24 hours: 89% PECAM blocked vs. 72% control; 72 hours: 69% PECAM blocked vs. 51% control). This correlated with a decrease in infarct volume. These findings suggest that TEM, in the setting of I/RI in the cerebrovascular, occurs primarily at the cortical surface. The reduction of stroke size with PECAM blockade suggests that infiltrating PMNs may exacerbate I/RI and indicate the potential therapeutic benefit of regulating the timing and pattern of leukocyte infiltration after stroke.

Mechanistic Insights into Macrophages Regulation of Neutrophil Transendothelial Migration in Inflamed Mucosa

Rapid neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires crossing of the vascular wall. PMN transendothelial migration (TEM) involves several well‐studied sequential adhesive interactions with vascular ECs, however what initiates or terminates this process is not well‐understood. Our findings identified a new mechanism where gut interstitial macrophages, which are rapidly recruited towards the vascular wall in response to inflammatory cues, were found to locally prime endothelial cells (ECs) responses to regulate PMN TEM. Using real‐time intravital microscopy (IVM) on lipopolysaccharides (LPS)‐inflamed intestines in anesthetized CX3CR1‐EGFP macrophage‐reporter mice, complemented by whole‐mount tissue imaging we demonstrate that macrophage presence was critical for the induction of PMN‐ECs adhesive interactions and subsequent PMN recruitment and accumulation in the intestinal mucosa. Anti CSFR‐1 antibody‐based macrophage depletion in the lamina propria and at the vessel wall significantly reduced PMN adhesion and TEM in inflamed intestines. We further observed that macrophages at the vessel wall localized specifically to regions of high ICAM‐1 intensity and their removal resulted in elimination of the ICAM‐1 “hot spots”, overall lowering the ECs ICAM‐1 expression. Mechanistically, using murine/human ECs‐macrophage and PMN co‐cultures we established that activated macrophages elevate PMN adhesion and TEM via TNFα‐dependent upregulation of ECs ICAM‐1. Antibody‐mediated neutralization of TNFα in macrophage co‐cultures with ECs and/or PMNs suppressed ICAM‐1 upregulation, decreasing PMN TEM. Further in vivo imaging studies of inflamed gut revealed high TNFα expression in macrophages and specific expression of TNFa receptor type II (TNFR II) but not type I in intestinal ECs. The use of bone marrow chimeras with TNFα knockout macrophages further confirmed the novel role of macrophages‐TNFα in regulating ECs adhesion molecule expression and PMN TEM. As such, our findings identify new, clinically relevant mechanism by which macrophages regulate PMN trafficking in inflamed mucosa.

Activation of brain endothelium by Escherichia coli K1 virulence factor cglD promotes polymorphonuclear leukocyte transendothelial migration

Escherichia coli K1 is the most common Gram-negative bacteria causing neonatal meningitis. Polymorphonuclear leukocyte (PMN) transmigration across the blood-brain barrier (BBB) is the hallmark of bacterial meningitis. Reportedly, the deletion of virulence factor cglD (E44:ΔcglD) from E44 is responsible for a less efficient PMN transendothelial migration ability. In the present study, we found that complementation of the cglD gene into E44:ΔcglD mutant strain might restore the PMN count and myeloperoxidase level in a neonatal mouse meningitis. Using human brain microvascular endothelial cells (HBMECs), the main model of the BBB in vitro, we found that E44:ΔcglD mutant strain induced a less efficient PMN adhesion to HBMECs and down-regulated chemokines CXCL1, CXCL6 and CXCL8 and adhesion molecule E-selectin, compared with the E44 strain. Complementation of cglD restored the PMN adhesion to HBMECs and the level of these proteins. E44:ΔcglD mutant strain also induced a less efficient NF-κB pathway activation in HBMECs and reduced the soluble p65 (sp65) level in the cerebral spinal fluid of newborn mice, compared with the E44 strain. Complementation of cglD restored the NF-κB pathway activation and increased the sp65 levels. This suggests that cglD in E44 contributes to NF-κB pathway activation in the brain endothelium to promote PMN adhesion to HBMECs and transendothelial migration. Our identified novel requirement of cglD for immune activation and subsequent PMN entry into the central nervous system suggests that therapies directed at neutralising this molecule will be beneficial in preventing bacterial meningitis progression.

In vivo imaging reveals unique neutrophil transendothelial migration patterns in inflamed intestines.

Neutrophil (PMN) infiltration of the intestinal mucosa is a hallmark of gastrointestinal inflammation, with significant implications for host defense, injury and repair. However, phenotypic and mechanistic aspects of PMN recruitment in inflamed intestines have not been explored in vivo. Using novel epithelial/PMN fluorescence reporter mice, advanced intravital imaging and 3D reconstruction analysis, we mapped the microvasculature architecture across the intestinal layers and determined that in response to Sa/mone//a/endotoxin-induced inflammation, PMN transendothelial migration (TEM) was restricted to submucosal vessels. PMN TEM was not observed in villus or crypt vessels, proximal to the epithelium that underlies the intestinal lumen, and was partially dependent on (C-X-C motif) ligands 1 (CXCL1) and 2 (CXCL2) expression, which was found to be elevated in the submucosa layer. Restricted PMN extravasation at the submucosa and subsequent PMN interstitial migration may serve as a novel regulatory step of PMN effector function and recruitment to the luminal space in inflamed intestines.

Carbon Monoxide–Releasing Molecule-401 Suppresses Polymorphonuclear Leukocyte Migratory Potential by Modulating F-Actin Dynamics

PMN co-cultures were perfused for additional 15 minutes with PMN-free medium containing CORM-401/inactive CORM-401. The experiments were videorecorded (phase-contrast microscopy), and PMN adhesion/migration were assessed off-line. In parallel, CORM-401-dependent modulation of PMN chemotaxis, F-actin expression/distribution, and actin-regulating pathways [eg, p21-activated protein kinases (PAK1/2) and extracellular signal-regulated kinase (ERK)/C-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK)] were assessed in response to N-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation. Pretreating PMNwith CORM-401 did not suppress PMN adhesion to HUVEC, but significantly reduced PMN transendothelial migration (P<0.0001) and fMLP-induced PMN chemotaxis (ie, migration directionality and velocity). These changes were associated with CORM-401-dependent suppression of F-actin levels/cellular distributionand fMLP-induced phosphorylation of PAK1/2 and ERK/JNK MAPK (P<0.05). CORM-401 had no effect on p38 MAPK activation. In summary, this study demonstrates, for the first time, CORM-401-dependent suppression of neutrophilmigratory potential associated with modulation of PAK1/2 and ERK/JNK MAPK signaling and F-actin dynamics.

Minocycline affects human neutrophil respiratory burst and transendothelial migration.

This study aimed at investigating the in vitro activity of minocycline and doxycycline on human polymorphonuclear (h-PMN) cell function. h-PMNs were isolated from whole venous blood of healthy subjects; PMN oxidative burst was measured by monitoring ROS-induced oxidation of luminol and transendothelial migration was studied by measuring PMN migration through a monolayer of human umbilical vein endothelial cells. Differences between multiple groups were determined by ANOVA followed by Tukey's multiple comparison test; Student's t test for unpaired data for two groups. Minocycline (1-300µM) concentration dependently and significantly inhibited oxidative burst of h-PMNs stimulated with 100nM fMLP. Ten micromolar concentrations, which are superimposable to C max following a standard oral dose of minocycline, promoted a 29.8±4% inhibition of respiratory burst (P<0.001; n=6). Doxycycline inhibited ROS production with a lesser extent and at higher concentrations. 10-100µM minocycline impaired PMN transendothelial migration, with maximal effect at 100µM (42.5±7%, inhibition, n=5, P<0.001). These results added new insight into anti-inflammatory effects of minocycline exerted on innate immune h-PMN cell function.

CD47 contributes to neutrophil recruitment (146.2)

Leukocyte recruitment plays a key role in an inflammatory response. CD47 associates “in cis” with β1, β2 and β3 integrins. CD47 also interacts “in trans” with Signal Regulatory Proteins (SIRP‐α and ‐γ) and thrombospondin (TSP). CD47 plays an important but incompletely understood role in the innate and adaptive immune responses. Neutrophil recruitment in CD47KO mice is reduced in models of peritonitis, lung injury and dermal air pouch inflammation, but the exact contribution(s) of CD47 to neutrophil (PMN) extravasation is unclear. Here we report that in the TNF‐α‐stimulated cremaster muscle microcirculation, PMN rolling velocity is elevated compared to WT (17.8±1.5μm/s vs. 14.3±1.0μm/s, respectively; p&lt;0.05), whereas there was no difference between WT and CD47KO mice in CXCL1‐induced arrest. Also, PMN transendothelial migration (TEM) is reduced by 30% in CD47KO mice compared to WT mice (p&lt;0.05). In vitro flow chamber assays demonstrate reduced adhesion (30% reduction vs. WT; p&lt;0.05) and TEM (60% reduction vs. WT; p&lt;0.05) of CD47KO PMN compared to WT PMN across WT TNF‐activated murine endothelium. We have previously reported that CD47 physically interacts with β2 integrins and regulates integrin affinity on T cells (Azcutia et al., Mol Biol Cell 2013). These previous observations and our current results suggest that CD47 plays a role in PMN recruitment through the regulation of β2 integrin adhesive function.Grant Funding Source: Supported by NIH HL‐36028 and AHA fellowship 11POST7730055.

ICAM-1–activated Src and eNOS signaling increase endothelial cell surface PECAM-1 adhesivity and neutrophil transmigration

AbstractPolymorphonuclear neutrophil (PMN) extravasation requires selectin-mediated tethering, intercellular adhesion molecule-1 (ICAM-1)–dependent firm adhesion, and platelet/endothelial cell adhesion molecule 1 (PECAM-1)–mediated transendothelial migration. An important unanswered question is whether ICAM-1–activated signaling contributes to PMN transmigration mediated by PECAM-1. We tested this concept and the roles of endothelial nitric oxide synthase (eNOS) and Src activated by PMN ligation of ICAM-1 in mediating PECAM-1–dependent PMN transmigration. We observed that lung PMN infiltration in vivo induced in carrageenan-injected WT mice was significantly reduced in ICAM-1−/− and eNOS−/− mice. Crosslinking WT mouse ICAM-1 expressed in human endothelial cells (ECs), but not the phospho-defective Tyr518Phe ICAM-1 mutant, induced SHP-2–dependent Src Tyr530 dephosphorylation that resulted in Src activation. ICAM-1 activation also stimulated phosphorylation of Akt (p-Ser473) and eNOS (p-Ser1177), thereby increasing NO production. PMN migration across EC monolayers was abolished in cells expressing the Tyr518Phe ICAM-1 mutant or by pretreatment with either the Src inhibitor PP2 or eNOS inhibitor L-NAME. Importantly, phospho–ICAM-1 induction of Src signaling induced PECAM-1 Tyr686 phosphorylation and increased EC surface anti–PECAM-1 mAb-binding activity. These results collectively show that ICAM-1–activated Src and eNOS signaling sequentially induce PECAM-1–mediated PMN transendothelial migration. Both Src and eNOS inhibition may be important therapeutic targets to prevent or limit vascular inflammation.

Febrile-Range Hyperthermia Modifies Endothelial and Neutrophilic Functions to Promote Extravasation

Acute respiratory distress syndrome (ARDS) is a neutrophil (polymorphonuclear leukocyte; PMN)-driven lung injury that is associated with fever and heat-stroke, and involves approximately 40% mortality. In murine models of acute lung injury (ALI), febrile-range hyperthermia (FRH) enhanced PMN accumulation, vascular permeability, and epithelial injury, in part by augmenting pulmonary cysteine-x-cysteine (CXC) chemokine expression. To determine whether FRH increases chemokine responsiveness within the lung, we used in vivo and in vitro models that bypass the endogenous generation of chemokines. We measured PMN transalveolar migration (TAM) in mice after intratracheal instillations of the human CXC chemokine IL-8 in vivo, and of IL-8-directed PMN transendothelial migration (TEM) through human lung microvascular endothelial cell (HMVEC-L) monolayers in vitro. Pre-exposure to FRH increased in vivo IL-8-directed PMN TAM by 23.5-fold and in vitro TEM by 7-fold. Adoptive PMN transfer demonstrated that enhanced PMN TAM required both PMN donors and recipients to be exposed to FRH, suggesting interdependent effects on PMNs and endothelium. FRH exposure caused the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase in lung homogenates and circulating PMNs, with an associated increase in HSP27 phosphorylation and stress-fiber formation. The inhibition of these signaling pathways with U0126 and SB203580 blocked the effects of FRH on PMN extravasation in vivo and in vitro. Collectively, these results (1) demonstrate that FRH augments chemokine-directed PMN extravasation through direct effects on endothelium and PMNs, (2) identify ERK and p38 signaling pathways in the effect, and (3) underscore the complex effects of physiologic temperature change on innate immune function and its potential consequences for lung injury.

Intravenous immunoglobulin G (IVIG) inhibits IL-1- and TNF-α-dependent, but not chemotactic-factor-stimulated, neutrophil transendothelial migration

High-dose intravenous immunoglobulin (IVIG) has anti-inflammatory effects via incompletely understood mechanisms. By investigating whether IVIG might modulate neutrophil (PMN) recruitment, we observed that IVIG dose-dependently inhibited (by 30-50%) PMN transendothelial migration (TEM) across human umbilical vein endothelial cells (EC) stimulated with IL-1α, IL-1β, TNF-α or IL-1β+TNF-α. Inhibition required the presence of IVIG with the responding PMNs, was attributable to the F(ab)(2) portion and was unrelated to putative contaminants in IVIG. IVIG did not inhibit IL-1β- or TNF-α-induced increase of PMN adhesion to EC, nor did it affect C5a- or IL-8-induced PMN TEM across unstimulated EC. Effects of IVIG and F(ab)(2) fragments were not associated with PMN activation, assessed by CD62L shedding, CD11b upregulation or PMN shape. Thus, IVIG selectively inhibits PMN TEM across inflammatory-cytokine-stimulated - but not unstimulated - EC, perhaps contributing to therapeutic benefit in chronic inflammation with minimal impact on chemotactic-factor-induced PMN recruitment during acute infection.

The novel non‐depolarizing, normokalemic cardioplegia formulation based on the combination of adenosine‐lidocaine (Adenocaine™) exerts superior anti‐neutrophil effects by synergistic actions of its components

ObjectivesWe tested the hypothesis that the combination of adenosine and lidocaine (AL) synergistically attenuates neutrophil (PMN) functions.Methods1. Superoxide anion (O2‐) generation, 2. PMN adherence, 3. Transmigration. Group differences (one‐way ANOVA) were accepted at P value&lt;0.05.ResultsThe isolated porcine PMNs released superoxide dismutase (0.1mg/ml)‐sensitive O2‐ in a burst pattern upon activation. Both adenosine and lidocaine alone inhibited O2‐ production in a dose‐dependent pattern. AL further inhibited O2‐ generation in a synergistic manner. In addition, adenosine (0.1–10 μM) inhibited O2‐ generation by cytochalasin B (priming), whereas lidocaine (1–100 μM) inhibited O2‐ release stimulated by both cytochalasin B and fMLP (activation). Both adenosine and lidocaine alone inhibited PAF‐induced PMN adherence to endothelial cells. Adenosine (50μM) and lidocaine (100 μM) decreased adherence to 71.6±5.4% (n=8, P&lt;0.05) and 73.2±8.1% (n=7, P&lt;0.05) of stimulated control, respectively. AL further suppressed adherence to 47.2±4.3% (n=7, P&lt;0.05). Adenosine (100μM) reduced PMN transendothelial migration to 80.1±6.7% of control (n=6, P&lt;0.05), whereas lidocaine (100μM) showed no significant inhibition. AL did not further decrease transmigration.ConclusionsAL suppresses PMN O2‐ generation and adherence in a synergistic manner relative to adenosine or lidocaine alone.

CORM‐3‐derived CO attenuates oxidative stress in vascular endothelial cells in SOD‐activity independent manner

Our previous studies indicate that CO derived from CO‐releasing molecule, CORM‐3, while suppressing PMN transendothelial migration at the same time amplifies oxidative stress in PMN, whereas oxidative stress in vascular endothelial cells (HUVEC) is effectively suppressed by CORM‐3. However, the mechanisms of such opposing effects of CO with respect to ROS production are unknown.In this study we assessed the effects of CORM‐3‐derived CO on modulation of the antioxidant enzyme, superoxide dismutase (SOD), activity in an in vitro model of endotoxemia. To this end, HUVEC were stimulated with LPS (1μg/ml) for 1–4h in the presence or absence of CORM‐3 (100μM) and assessed for production of ROS, activation of NFκB and expression of adhesion molecule ICAM‐1. In parallel, SOD activity in cell lysates (cell‐associated SOD) and supernatants (soluble SOD) were also assessed.The obtained results indicate that LPS‐induced increase in ROS production, activation of NFκB and ICAM‐1 expression were effectively reduced by CORM‐3. Interestingly, CORM‐3‐dependent suppression of ROS production was not associated with increased SOD activity. On the contrary, CORM‐derived CO attenuated SOD activity in both, cell lysates and supernatants, and in an assay employing purified SOD1 enzyme, indicating that CORM‐3‐derived CO negatively regulates SOD activity in cultured vascular endothelial cells (HSFO‐NA6171; IRF‐025‐09).

CORM-3-derived CO modulates polymorphonuclear leukocyte migration across the vascular endothelium by reducing levels of cell surface-bound elastase

Recently, it has been shown that carbon monoxide (CO)-releasing molecule (CORM)-released CO can suppress inflammation. In this study, we assessed the effects and potential mechanisms of a ruthenium-based water-soluble CO carrier [tricarbonylchloroglycinate-ruthenium(II) (CORM-3)] in the modulation of polymorphonuclear leukocyte (PMN) inflammatory responses in an experimental model of sepsis. Sepsis in mice was induced by cecal ligation and puncture. CORM-3 (3 mg/kg iv) was administered 15 min after the induction of cecal ligation and puncture. PMN accumulation in the lung (myeloperoxidase assay), bronchoalveolar lavage (BAL) fluid, and lung vascular permeability (protein content in BAL fluid) were assessed 6 h later. In in vitro experiments, human PMNs were primed with LPS (10 ng/ml) and subsequently stimulated with formyl-methionyl-leucylphenylalanine (fMLP; 100 nM). PMN production of ROS (L-012/dihydrorhodamine-123 oxidation), degranulation (release of elastase), and PMN rolling, adhesion, and migration to/across human umbilical vein endothelial cells (HUVECs) were assessed in the presence or absence of CORM-3 (1-100 muM). The obtained results indicated that systemically administered CORM-3 attenuates PMN accumulation and vascular permeability in the septic lung. Surprisingly, in in vitro experiments, treatment of PMNs with CORM-3 further augmented LPS/fMLP-induced ROS production and the release of elastase. The latter effects, however, were accompanied by an inability of PMNs to mobilize elastase to the cell surface (plasma membrane), an event required for efficient PMN transendothelial migration. The CORM-3-induced decrease in cell surface levels of elastase was followed by decreased PMN rolling/adhesion to HUVECs and complete prevention of PMN migration across HUVECs. In contrast, treatment of HUVECs with CORM-3 had no effect on PMN transendothelial migration. Taken together, these findings indicate that, in sepsis, CORM3-released CO, while further amplifying ROS production and degranulation of PMNs, concurrently reduces the levels of cell surface-bound elastase, which contributes to suppressed PMN transendothelial migration.

Francisella tularensisdirectly interacts with the endothelium and recruits neutrophils with a blunted inflammatory phenotype

Francisella tularensis, the causative agent of tularemia, is a highly virulent organism, especially when exposure occurs by inhalation. Recent data suggest that Francisella interacts directly with alveolar epithelial cells. Although F. tularensis causes septicemia and can live extracellularly in a murine infection model, there is little information about the role of the vascular endothelium in the host response. We hypothesized that F. tularensis would interact with pulmonary endothelial cells as a prerequisite to the clinically observed recruitment of neutrophils to the lung. Using an in vitro Transwell model system, we studied interactions between F. tularensis live vaccine strain (Ft LVS) and a pulmonary microvascular endothelial cell (PMVEC) monolayer. Organisms invaded the endothelium and were visualized within individual endothelial cells by confocal microscopy. Although these bacteria-endothelial cell interactions did not elicit production of the proinflammatory chemokines, polymorphonuclear leukocytes (PMN) were stimulated to transmigrate across the endothelium in response to Ft LVS. Moreover, transendothelial migration altered the phenotype of recruited PMN; i.e., the capacity of these PMN to activate NADPH oxidase and release elastase in response to subsequent stimulation was reduced compared with PMN that traversed PMVEC in response to Streptococcus pneumoniae. The blunting of PMN responsiveness required PMN transendothelial migration but did not require PMN uptake of Ft LVS, was not dependent on the presence of serum-derived factors, and was not reproduced by Ft LVS-conditioned medium. We speculate that the capacity of Ft LVS-stimulated PMVEC to support transendothelial migration of PMN without triggering release of IL-8 and monocyte chemotactic protein-1 and to suppress the responsiveness of transmigrated PMN to subsequent stimulation could contribute to the dramatic virulence during inhalational challenge with Francisella.

NADPH oxidase contributes to conversion of cardiac myocytes to a proinflammatory phenotype in sepsis

It has been reported that polymorphonuclear leukocyte (PMN) infiltration into the myocardial interstitium is involved in sepsis-induced myocardial dysfunction. The aim of this study was to evaluate the role of NADPH oxidase in the sepsis-induced conversion of cardiomyocytes to a proinflammatory phenotype. Using an in vitro approach we evaluated the role of NADPH oxidase in cardiomyocyte CXC chemokine production and its ability to promote PMN transendothelial migration under septic conditions. Treatment of cardiac myocytes with septic plasma (1) activated NADPH oxidase (p47phox phosphorylation) and increased its activity (O 2 − production) and (2) converted them to a proinflammatory phenotype; both effects were prevented by blockade of NADPH oxidase. NF-κB nuclear translocation was increased in cardiomyocytes conditioned with septic plasma, a response prevented by blockade of NADPH oxidase. The increase in NF-κB activation/translocation was associated with phosphorylation of both IKK and the p65 subunit of NF-κB. Blockade of NADPH oxidase prevented phosphorylation of IKK, but not p65. Blockade approaches indicated that p38 MAP kinase (previously implicated in NF-κB activation) did not play a role in the NADPH oxidase pathway, either upstream or downstream. Collectively, the results of this study and those of previous reports indicate that the conversion of cardiomyocytes to a proinflammatory phenotype in sepsis involves two distinct pathways: NADPH oxidase-mediated phosphorylation of IKK and p38 MAP kinase-mediated phosphorylation of p65.

Alveolar Macrophages from Septic Mice Promote Polymorphonuclear Leukocyte Transendothelial Migration via an Endothelial Cell Src Kinase/NADPH Oxidase Pathway

Alveolar macrophages (AMphi) have been implicated in the polymorphonuclear leukocyte (PMN) recruitment to the lungs during sepsis. Using an in vivo murine model of sepsis (feces in the peritoneum), we show that peritonitis leads to increased activation of AMphi and PMN migration into pulmonary alveoli. To assess cellular mechanisms, an in vitro construct of the pulmonary vascular-interstitial interface (murine AMphi, pulmonary endothelial cells, and PMN) and a chimera approach were used. Using immunologic (Abs) and genetic blockade (CXCR2-deficient AMphi), we show that CXC chemokines in septic plasma are responsible for the activation of AMphi. The activated AMphi can promote PMN transendothelial migration, even against a concentration gradient of septic plasma, by generating platelet-activating factor and H(2)O(2). Platelet-activating factor/H(2)O(2) induce an oxidant stress in the adjacent endothelial cells, an event that appears to be a prerequisite for PMN transendothelial migration, since PMN migration is abrogated across Cu/Zn-superoxide dismutase overexpressing endothelial cells. Using gp91-deficient endothelial cells, we show that NADPH oxidase plays an important role in the AMphi-induced PMN transendothelial migration. Pharmacologic/small interfering RNA blockade of Src kinase inhibits AMphi-induced endothelial NADPH oxidase activation and PMN migration. Collectively, our findings indicate that the PMN transendothelial migration induced by septic AMphi is dependent on the generation of superoxide in endothelial cells via the Src kinase/NADPH oxidase signaling pathway.

ClC-3 and IClswell are Required for Normal Neutrophil Chemotaxis and Shape Change

Polymorphonuclear leukocytes undergo directed movement to sites of infection, a complex process known as chemotaxis. Extension of the polymorphonuclear leukocyte (PMN) leading edge toward a chemoattractant in association with uropod retraction must involve a coordinated increase/decrease in membrane, redistribution of cell volume, or both. Deficits in PMN phagocytosis and trans-endothelial migration, both highly motile PMN functions, suggested that the anion transporters, ClC-3 and ICl(swell), are involved in cell motility and shape change ( Moreland, J. G., Davis, A. P., Bailey, G., Nauseef, W. M., and Lamb, F. S. (2006) J. Biol. Chem. 281, 12277-12288 ). We hypothesized that ClC-3 and ICl(swell) are required for normal PMN chemotaxis through regulation of cell volume and shape change. Using complementary chemotaxis assays, EZ-TAXIScantrade mark and dynamic imaging analysis software, we analyzed the directed cell movement and morphology of PMNs lacking normal anion transporter function. Murine Clcn3(-/-) PMNs and human PMNs treated with anion transporter inhibitors demonstrated impaired chemotaxis in response to formyl peptide. This included decreased cell velocity and failure to undergo normal cycles of elongation and retraction. Impaired chemotaxis was not due to a diminished number of formyl peptide receptors in either murine or human PMNs, as measured by flow cytometry. Murine Clcn3(-/-) and Clcn3(+/+) PMNs demonstrated a similar regulatory volume decrease, indicating that the ICl(swell) response to hypotonic challenge was intact in these cells. We further demonstrated that ICl(swell) is essential for shape change during human PMN chemotaxis. We speculate that ClC-3 and ICl(swell) have unique roles in regulation of PMN chemotaxis; ICl(swell) through direct effects on PMN volume and ClC-3 through regulation of ICl(swell).

Points of Control Exerted along the Macrophage-Endothelial Cell-Polymorphonuclear Neutrophil Axis by PECAM-1 in the Innate Immune Response of Acute Colonic Inflammation

PECAM-1 is expressed on endothelial cells and leukocytes. Its extracellular domain has been implicated in leukocyte diapedesis. In this study, we used PECAM-1(-/-) mice and relevant cells derived from them to assess the role of PECAM-1 in an experimental model of acute colonic inflammation with a predominant innate immune response, i.e., 2,4,6-trinitrobenzine sulfonic acid (TNBS). Using chimeric approaches, we addressed the points of control exerted by PECAM-1 along the macrophage-endothelial cell-polymorphonuclear neutrophil (PMN) axis. In vivo, TNBS-induced colitis was ameliorated in PECAM-1(-/-) mice, an event attributed to PECAM-1 on hematopoietic cells rather than to PECAM-1 on endothelial cells. The in vivo innate immune response was mimicked in vitro by using a construct of the vascular-interstitial interface, i.e., PMN transendothelial migration was induced by colonic lavage fluid (CLF) from TNBS mice or macrophages (MPhi) challenged with CLF. Using the construct, we confirmed that endothelial cell PECAM-1 does not play a role in PMN transendothelial migration. Although MPhi activation (NF-kappaB nuclear binding) and function (keratinocyte-derived chemokine production) induced by CLF was diminished in PECAM-1(-/-) MPhi, this did not affect their ability to promote PMN transendothelial migration. By contrast, PECAM-1(-/-) PMN did not adhere to or migrate across endothelial cell monolayers in response to CLF. Further, as compared with PECAM-1(+/+) PMN, PECAM-1(-/-) PMN were less effective in orientating their CXCR2 receptors (polarization) in the direction of a chemotactic gradient. Collectively, our findings indicate that PECAM-1 modulation of PMN function (at a step before diapedesis) most likely contributes to the inflammation in a colitis model with a strong innate immune component.

Polymorphonuclear leukocyte (PMN) migration across vascular endothelial cells (EC) in the absence or presence of IL‐1β differentially regulate expression of EC adhesion molecules ICAM‐1 and E‐selectin

Our findings indicate that PMN migration across activated EC results in down‐regulation of EC nuclear transcription factor, NFκB. However, the subsequent functional consequences are largely unknown.In this study we assessed the effects of PMN transendothelial migration on NFκB‐dependent expression of EC adhesion molecules, ICAM‐1 and E‐selectin during inflammation.To this end human endothelial cells (HUVEC) were grown in Traswell inserts and stimulated with IL‐1β (1ng/ml; 4 hrs). Subsequently, PMN were added and allowed to migrate across HUVEC in response to fMLP (10−8M) chemotactic gradient in the absence (i.e. across washed HUVEC) or presence of IL‐1β. ICAM‐1 and E‐selectin gene expression in EC was assessed by RT‐PCR after 4 or 24 hrs following PMN migration.The obtained results indicate that migration of PMN across IL‐1β treated and subsequently washed HUVEC results in a marked down‐regulation of IL‐1 β‐induced ICAM‐1, and to a lesser degree, E‐selectin gene expression as assessed 4 and 24 hrs following PMN migration. On the contrary, migration of PMN across activated HUVEC in the presence of IL‐1β results in a further increase in both, ICAM‐1 and E‐selectin expression.Taken together these findings indicate that migrating PMN differentially modulate expression of EC adhesion molecules during inflammation and can offer EC with both, anti‐ and pro‐inflammatory stimuli (HSFO‐NA5580/NA6171)