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Essentially all Staphylococcus aureus (S. aureus) bacteria that gain access to the circulation are plucked out of the bloodstream by the intravascular macrophages of the liver--the Kupffer cells. It is also thought that these bacteria are disseminated via the bloodstream to other organs. Our data show that S. aureus inside Kupffer cells grew and escaped across the mesothelium into the peritoneal cavity and immediately infected GATA-binding factor 6-positive (GATA6+) peritoneal cavity macrophages. These macrophages provided a haven for S. aureus, thereby delaying the neutrophilic response in the peritoneum by 48 hours and allowing dissemination to various peritoneal and retroperitoneal organs including the kidneys. In mice deficient in GATA6+ peritoneal macrophages, neutrophils infiltrated more robustly and reduced S. aureus dissemination. Antibiotics administered i.v. did not prevent dissemination into the peritoneum or to the kidneys, whereas peritoneal administration of vancomycin (particularly liposomal vancomycin with optimized intracellular penetrance capacity) reduced kidney infection and mortality, even when administered 24 hours after infection. These data indicate that GATA6+ macrophages within the peritoneal cavity are a conduit of dissemination for i.v. S. aureus, and changing the route of antibiotic delivery could provide a more effective treatment for patients with peritonitis- associated bacterial sepsis., Introduction In humans, Staphylococcus aureus (S. aureus) bacteremia is the most common serious bacterial blood infection globally. In the United Kingdom alone, approximately 12,500 cases each year are reported with [...]

Neutrophils are the most abundant immune cells in humans and serve as first responders to a myriad of host perturbations. Equipped with a plethora of antimicrobial molecules, neutrophils invade sites of inflammation to eradicate pathogens and clear debris. Traditionally, neutrophils were thought to cause collateral tissue damage before dying at the site. However, the presence of neutrophil infiltration into sterile injuries (in the absence of infections) suggests additional roles for these cells. Now, the view of neutrophils as indiscriminate killers seems to be changing as evolving evidence suggests that neutrophils actively orchestrate resolution of inflammation and contribute to tissue repair. Novel concepts include the idea that neutrophils are key to revascularization and subsequently reverse-transmigrate back to the vasculature, actively leaving sites of tissue damage to re-home to functional niches in the lung and bone marrow. This Review scrutinizes the role of neutrophils in tissue damage and repair, discussing recent findings and raising unresolved questions around this intriguing immune cell., Introduction Neutrophils are the most abundant immune cells, constituting about 60% to 70% of all leukocytes in human blood (1, 2). Neutrophils act as the first responders of the innate [...]

Radiographic contrast agents cause acute kidney injury (AKI), yet the underlying pathogenesis is poorly understood. Nod-like receptor pyrin containing 3-deficient (Nlrp3-deficient) mice displayed reduced epithelial cell injury and inflammation in the kidney in a model of contrast-induced AKI (CI-AKI). Unexpectedly, contrast agents directly induced tubular epithelial cell death in vitro that was not dependent on Nlrp3. Rather, contrast agents activated the canonical Nlrp3 inflammasome in macrophages. Intravital microscopy revealed diatrizoate (DTA) uptake within minutes in perivascular [CX.sub.3][CR1.sup.+] resident phagocytes in the kidney. Following rapid filtration into the tubular luminal space, DTA was reabsorbed and concentrated in tubular epithelial cells via the brush border enzyme dipeptidase-1 in volume-depleted but not euvolemic mice. [LysM-GFP.sup.+] macrophages recruited to the kidney interstitial space ingested contrast material transported from the urine via direct interactions with tubules. CI-AKI was dependent on resident renal phagocytes, IL-1, leukocyte recruitment, and dipeptidase-1. Levels of the inflammasome-related urinary biomarkers IL-18 and caspase-1 were increased immediately following contrast administration in patients undergoing coronary angiography, consistent with the acute renal effects observed in mice. Taken together, these data show that CI-AKI is a multistep process that involves immune surveillance by resident and infiltrating renal phagocytes, Nlrp3-dependent inflammation, and the tubular reabsorption of contrast via dipeptidase-1., Introduction Contrast-induced acute kidney injury (CI-AKI) is caused by iodinated contrast media for diagnostic imaging (1). Most patients with normal kidney function who receive contrast will not experience any renal [...]

Pseudomonas aeruginosa is a major cause of severe infections that lead to bacteremia and high patient mortality. P. aeruginosa has evolved numerous evasion and subversion mechanisms that work in concert to overcome immune recognition and effector functions in hospitalized and immunosuppressed individuals. Here, we have used multilaser spinning-disk intravital microscopy to monitor the blood-borne stage in a murine bacteremic model of P. aeruginosa infection. P. aeruginosa adhered avidly to lung vasculature, where patrolling neutrophils and other immune cells were virtually blind to the pathogen's presence. This cloaking phenomenon was attributed to expression of Psl exopolysaccharide. Although an antiPsl mAb activated complement and enhanced neutrophil recognition of P. aeruginosa, neutrophil-mediated clearance of the pathogen was suboptimal owing to a second subversion mechanism, namely the type 3 secretion (T3S) injectisome. Indeed, T3S prevented phagosome acidification and resisted killing inside these compartments. Antibody-mediated inhibition of the T3S protein PcrV did not enhance bacterial phagocytosis but did enhance killing of the few bacteria ingested by neutrophils. A bispecific mAb targeting both Psl and PcrV enhanced neutrophil uptake of P. aeruginosa and also greatly increased inhibition of T3S function, allowing for phagosome acidification and bacterial killing. These data highlight the need to block multiple evasion and subversion mechanisms in tandem to kill P. aeruginosa., Introduction Pseudomonas aeruginosa is an important opportunistic bacterial pathogen that causes life-threatening infections in critically ill and immunocompromised individuals. It is one of the most common causes of Gram-negative pneumonia [...]

The production of neutrophil extracellular traps (NETs) is a process that enables neutrophils to help catch and kill bacteria. However, increasing evidence suggests that this process might also occur in noninfectious, sterile inflammation. In this Review, we describe the role of NETosis in autoimmunity, coagulation, acute injuries and cancer, and discuss NETs as potential therapeutic targets. Furthermore, we consider whether extracellular DNA is always detrimental in sterile inflammation and whether the source is always NETs., Author(s): Selina K Jorch [1]; Paul Kubes (corresponding author) [1, 2] Neutrophil extracellular traps (NETs) are structures of chromatin filaments coated with histones, proteases and granular and cytosolic proteins, and [...]

The use of monoclonal antibodies (mAbs) as therapeutic tools has increased dramatically in the last decade and is now one of the mainstream strategies to treat cancer. Nonetheless, it is still not completely understood how mAbs mediate tumor cell elimination or the effector cells that are involved. Using intravital microscopy, we found that antibody-dependent phagocytosis (ADPh) by macrophages is a prominent mechanism for removal of tumor cells from the circulation in a murine tumor cell opsonization model. Tumor cells were rapidly recognized and arrested by liver macrophages (Kupffer cells). In the absence of mAbs, Kupffer cells sampled tumor cells; however, this sampling was not sufficient for elimination. By contrast, antitumor mAb treatment resulted in rapid phagocytosis of tumor cells by Kupffer cells that was dependent on the high-affinity IgG-binding Fc receptor (FcγRI) and the low-affinity IgG-binding Fc receptor (FcγRIV). Uptake and intracellular degradation were independent of reactive oxygen or nitrogen species production. Importantly, ADPh prevented the development of liver metastases. Tumor cell capture and therapeutic efficacy were lost after Kupffer cell depletion. Our data indicate that macrophages play a prominent role in mAb-mediated eradication of tumor cells. These findings may help to optimize mAb therapeutic strategies for patients with cancer by helping us to aim to enhance macrophage recruitment and activity., Introduction Therapeutic monoclonal antibodies (mAbs), which can be designed to specifically interact with tumor-associated antigens, represent a promising novel category of drugs for targeting malignancies in addition to chemotherapy or [...]

The majority of patients with cancer undergo at least one surgical procedure as part of their treatment. Severe postsurgical infection is associated with adverse oncologic outcomes; however, the mechanisms underlying [...]

Here we focus on how neutrophils have a key regulatory role in vascular inflammation. Recent studies using advanced imaging techniques have yielded new insights into the mechanisms by which neutrophils contribute to defense against bacterial infections and also against sterile injury. In these settings, neutrophils are recruited by various mechanisms depending on the situation. We also describe how these processes may be disrupted in systemic infections, with a particular emphasis on mouse models of sepsis. Neutrophils are often immobilized in the lungs and liver during systemic infections, and this immobilization may be a mechanism through which bacteria can evade the innate immune response or allow neutrophils to form neutrophil extracellular traps that trap and kill bacteria in blood. The platelet is also an important player in sepsis, and we describe how it collaborates with neutrophils in the formation of neutrophil extracellular traps., The neutrophil patrols the blood in continuous search of prey, which presents itself primarily in the form of bacteria or dead and dying host cells. Because more than 4 trillion [...]

Neutrophils are recruited from the brood to sites of sterile inflammation, where they contribute to wound healing but may also cause tissue damage. By using spinning disk confocal intravital microscopy, we examined the kinetics and molecular mechanisms of neutrophil recruitment to sites of focal hepatic necrosis in vivo. Adenosine triphosphate released from necrotic cells activated the Nlrp3 inflammasome to generate an inflammatory microenvironment that alerted circulating neutrophils to adhere within liver sinusoids. Subsequently, generation of an intravascular chemokine gradient directed neutrophil migration through healthy tissue toward foci of damage. Lastly, formyl-peptide signals released from necrotic cells guided neutrophils through nonperfused sinusoids into the injury. Thus, dynamic in vivo imaging revealed a multistep hierarchy of directional cues that guide neutrophil localization to sites of sterile inflammation. 10.1126/science.1195491

Infectious meningitis and encephalitis is caused by invasion of circulating pathogens into the brain. It is unknown how the circulating pathogens dynamically interact with brain endothelium under shear stress, leading to invasion into the brain. Here, using intravital microscopy, we have shown that Cryptococcus neoformans, a yeast pathogen that causes meningoencephalitis, stops suddenly in mouse brain capillaries of a similar or smaller diameter than the organism, in the same manner and with the same kinetics as polystyrene microspheres, without rolling and tethering to the endothelial surface. Trapping of the yeast pathogen in the mouse brain was not affected by viability or known virulence factors. After stopping in the brain, C. neoformans was seen to cross the capillary wall in real time. In contrast to trapping, viability, but not replication, was essential for the organism to cross the brain microvasculature. Using a knockout strain of C. neoformans, we demonstrated that transmigration into the mouse brain is urease dependent. To determine whether this could be amenable to therapy, we used the urease inhibitor flurofamide. Flurofamide ameliorated infection of the mouse brain by reducing transmigration into the brain. Together, these results suggest that C. neoformans is mechanically trapped in the brain capillary, which may not be amenable to pharmacotherapy, but actively transmigrates to the brain parenchyma with contributions from urease, suggesting that a therapeutic strategy aimed at inhibiting this enzyme could help prevent meningitis and encephalitis caused by C. neoformans infection., Introduction Infectious meningitis and encephalitis is a devastating disease associated with high mortality and morbidity throughout the world. For disease to develop, the circulating pathogen must be arrested in the [...]

Recognition of LPS by TLR4 on immune sentinel cells such as macrophages is thought to be key to the recruitment of neutrophils to sites of infection with Gram-negative bacteria. To explore whether endothelial TLR4 plays a role in this process, we engineered and imaged mice that expressed TLR4 exclusively on endothelium (known herein as [Endothelium.sup.TLR4] mice). Local administration of LPS into tissue induced comparable neutrophil recruitment in [Endothelium.sup.TLR4] and wild-type mice. Following systemic LPS or intraperitoneal E. coli administration, most neutrophils were sequestered in the lungs of wild-type mice and did not accumulate at primary sites of infection. In contrast, [Endothelium.sup.TLR4] mice showed reduced pulmonary capillary neutrophil sequestration over the first 24 hours; as a result, they mobilized neutrophils to primary sites of infection, cleared bacteria, and resisted a dose of E. coli that killed 50% of wild-type mice in the first 48 hours. In fact, the only defect we detected in [Endothelium.sup.TLR4] mice was a failure to accumulate neutrophils in the lungs following intratracheal administration of LPS; this response required TLR4 on bone marrow--derived immune cells. Therefore, endothelial TLR4 functions as the primary intravascular sentinel system for detection of bacteria, whereas bone marrow--derived immune cells are critical for pathogen detection at barrier sites. Nonendothelial TLR4 contributes to failure to accumulate neutrophils at primary infection sites in a disseminated systemic infection., Introduction Localized infection with Gram-negative bacteria activates the local microenvironment and causes the influx of neutrophils into the afflicted tissue. The neutrophils eradicate the pathogen without eliciting any notable systemic [...]

Staphylococcal superantigens are pyrogenic exotoxins that cause massive T cell activation leading to toxic shock syndrome and death. Despite the strong adaptive immune response induced by these toxins, infections by superantigen-producing staphylococci are very common clinical events. We hypothesized that this may be partly a result of staphylococcal strains having developed strategies that downregulate the T cell response to these toxins. Here we show that the human interleukin-2 response to staphylococcal superantigens is inhibited by the simultaneous presence of bacteria. Such a downregulatory effect is the result of peptidoglycan-embedded molecules binding to Toll-like receptor 2 and inducing interleukin-10 production and apoptosis of antigen-presenting cells. We corroborated these findings in vivo by showing substantial prevention of mortality after simultaneous administration of staphylococcal enterotoxin B with either heat-killed staphylococci or Staphylococcus aureus peptidoglycan in mouse models of superantigen-induced toxic shock syndrome., S. aureus is a common human commensal organism, present in the upper respiratory tract of 25-50% of the population (1). It is also one of the most frequent pathogens, being [...]

It has been known for many years that neutrophils and platelets participate in the pathogenesis of severe sepsis, but the inter-relationship between these players is completely unknown. We report several cellular events that led to enhanced trapping of bacteria in blood vessels: platelet TLR4 detected TLR4 ligands in blood and induced platelet binding to adherent neutrophils. This led to robust neutrophil activation and formation of neutrophil extracellular traps (NETs). Plasma from severely septic humans also induced TLR4-dependent platelet-neutrophil interactions, leading to the production of NETs. The NETs retained their integrity under flow conditions and ensnared bacteria within the vasculature. The entire event occurred primarily in the liver sinusoids and pulmonary capillaries, where NETs have the greatest capacity for bacterial trapping. We propose that platelet TLR4 is a threshold switch for this new bacterial trapping mechanism in severe sepsis., Author(s): Stephen R Clark [1, 6]; Adrienne C Ma [1, 6]; Samantha A Tavener [1]; Braedon McDonald [1]; Zahra Goodarzi [1]; Margaret M Kelly [1, 2]; Kamala D Patel [1, [...]

The inflammatory endothelial response to LPS is critical to the host's surviving a gram-negative bacterial infection. In this study we investigated whether human endothelial cells express the functional coreceptor for LPS, CD 14, and most importantly whether it is glycosylphosphatidylinositol (GPI) linked. We also examined whether plasma proteins could reconstitute an LPS response in CD14-inhibited endothelium. RT-PCR- and CD14-specific MAbs demonstrated CD14 expression on primary human umbilical vein endothelial cells (HUVEC) but not passaged HUVEC. The amino acid sequence of endothelial CD14 was 99% homologous to CD14 on monocytes. Endothelium responded to relatively low levels of LPS in the absence of plasma, and this was entirely dependent on CD14. Removal of GPI-linked proteins with phosphatidylinositol-phospholipase C prevented LPS detection and subsequent protein synthesis (E-selectin expression). Endothelial CD14 was sufficient to initiate functional leukocyte recruitment, an event inhibited by blocking its LPS binding epitope and also by removing CD14 from the endothelial surface. Plasma proteins restored only ~30% of the LPS response in CD14-inhibited endothelium. In conclusion, our results strongly support an important role for endothelial membrane CD14 in the activation of endothelium for leukocyte recruitment. human; human umbilical vein endothelial cells; inflammation; leukocyte recruitment; glycosylphosphatidylinositol

Recently we reported that Toll-like receptor 4 (TLR4)-positive immune cells of unknown identity were responsible for the LPS-induced depression of cardiac myocyte shortening. The aim of this study is to identify the TLR4-positive cell type that is responsible for the LPS-induced cardiac dysfunction. Neither neutrophil depletion alone nor mast cell deficiency had any impact on the impairment of myocyte shortening during LPS treatment. In contrast, LPS-treated, macrophage-deficient mice demonstrated a partial reduction in shortening compared with saline-treated, macrophage-deficient mice. Because the removal of macrophages could only partially restore myocyte shortening, we also investigated the effects of removing both neutrophils and macrophages on myocyte shortening. Interestingly, endotoxemic, neutrophil-depleted, and macrophage-deficient mice had completely restored myocyte shortening. Because both macrophages and neutrophils can produce nitric oxide (NO) and TNF-[alpha], we examined LPS-treated inducible NO synthase knockout (iNOSKO) mice and TNF receptor (TNFR)-deficient mice. Eliminating both TNFR1 and TNFR2 was required to restore myocyte shortening during LPS treatment, whereas iNOS deficiency had no effect. These data suggest that macrophages and to a lesser degree neutrophils cause cardiac impairment, presumably via TNF-[alpha]. neutrophils; mast cells; macrophages; endotoxemia; tumor necrosis factor-[alpha]

After an ischemic episode, reperfusion causes profound oxidative stress in the vasculature of the afflicted tissue/ organ. The dysregulated accumulation of reactive oxygen species (ROS), such as superoxide, has been closely linked to the production and release of proinflammatory mediators, a profound increase in adhesion molecule expression by the vascular endothelium, and infiltration of neutrophils during ischemia-reperfusion (I/R). Superoxide dismutase (SOD) has been shown to protect tissues and organs against I/R-induced injury; however, the drug had to be continuously perfused or kidneys had to be occluded to prevent clearance. We used intravital microscopy, a system that allowed us to visualize neutrophil-endothelial interactions within the mesenteric postcapillary venules of cats subjected to I/R and tested the hypothesis that I/R-induced neutrophil recruitment was inhibited by treatment with SOD2/3. SOD2/3 is a chimeric fusion gene product that contains the mature SOD2 as well as the COOH-terminal 'tail' of SOD3 and, unlike the three naturally occurring SODs (SOD1, SOD2, and SOD3), which bear a net negative charge at pH 7.4, SOD2/3 is positively charged and physiologically stable. Our results suggest that not only does SOD2/3 have a much greater efficacy in vivo than the native human SOD2, but its administration prevents I/R-induced neutrophil-endothelial cell interactions and microvascular dysfunction. Moreover, our data support the hypothesis that reactive oxidants mediate I/R-induced injury and that the chimeric recombinant SOD2/3 has the potential to be a therapeutic agent against this debilitating illness. superoxide dismutase; in vivo; endothelium

A hallmark feature of intestinal inflammation is the recruitment and extravasation of numerous cell types from the blood to the afflicted site. Much of what we know about the mechanisms of leukocyte recruitment to splanchnic organs comes from an extensive series of studies on neutrophils in the mesenteric microvasculature. In this themes article, we highlight the important findings from these experiments but also emphasize some of the limitations. In fact, there is a growing body of evidence that neutrophil recruitment may be quite different in the mesentery than in other splanchnic organs. For example, the molecular mechanisms underlying neutrophil recruitment into the liver are quite different than the mesentery and are dependent on the type of inflammatory disease. We also discuss the effect of modulating leukocyte recruitment to splanchnic organs in chronic inflammation and emphasize that the approaches that have been successful in acute inflammation may be less effective in such conditions as inflammatory bowel disease (IBD). One obvious reason for this observation is the growing body of evidence to suggest that the initiation and maintenance of IBD is, in part, due to dysregulated or inappropriately activated populations of infiltrating T lymphocyte subsets. Therefore, we also discuss some interesting new approaches to limiting lymphocyte recruitment into the inflamed intestine either by targeting T helper (Th)1 vs. Th2 lymphocytes or perhaps by allowing the recruitment of regulatory T cells. Inhibiting specific adhesion molecules or specific chemokine receptors may work in this regard. lymphocyte; intestine; liver; neutrophils; adhesion molecules

Thrombin-stimulated endothelium synthesizes numerous adhesion molecules to recruit leukocytes; however, it is unknown which intracellular pathways are responsible for this event. A recent report from our laboratory has shown that thrombin induces E-selectin expression and that blocking nuclear factor-[kappa]B (NF-[kappa]B) activity partially blocked both E-selectin expression (60%) and leukocyte recruitment. In this study, we systematically assessed the importance of p38 MAPK in thrombin-induced NF-[kappa]B activation and E-selectin-dependent leukocyte recruitment. Thrombin caused phosphorylation of p38 MAPK, its substrate ATF-2, and JNK MAPK, but not ERK MAPK. The p38 MAPK inhibitors, SKF86002 and SB-203580 only reduced ATF-2 activity. We treated human umbilical vein endothelial cells with SKF86002, 1 h before thrombin stimulation, and noted inhibition of NF-[kappa]B mobilization and complete inhibition of leukocyte rolling and adhesion in a laminar flow chamber. Significant inhibition of leukocyte recruitment and E-selectin expression was also observed with SB-203580. SKF86002 did not affect other systems, including tumor necrosis factor-[alpha]-induced E-selectin-dependent leukocyte recruitment. Moreover, thrombin-induced rapid mobilization of P-selectin from Weibel Palade bodies was not p38 MAPK dependent. These data suggest that thrombin induces p38 MAPK activation, which leads to NF-[kappa]B mobilization to the nucleus and causes the upregulation of E-selectin and subsequent leukocyte recruitment. endothelium; selectins; inflammation

Neutrophils must follow both endogenous and bacterial chemoattractant signals out of the vasculature and through the interstitium to arrive at a site of infection. By necessity, in the setting of multiple chemoattractants, the neutrophils must prioritize, favoring end target chemoattractants (e.g., fMLP and C5a) emanating from the site of infection over intermediary endogenous chemoattractants (e.g., IL-8 and LT[B.sub.4]) encountered en route to sites of infection. In this study, we propose a hierarchical model of two signaling pathways mediating the decision-making process of the neutrophils, which allows end target molecules to dominate over intermediary chemoattractants. In an under agarose assay, neutrophils predominantly migrated toward end target chemoattractants via p38 MAPK, whereas intermediary chemoattractant-induced migration was phosphoinositide 3-kinase (PI3K)/Akt dependent. When faced with competing gradients of end target and intermediary chemoattractants, Akt activation was significantly reduced within neutrophils, and the cells migrated preferentially toward end target chemoattractants even at 1/1,000th that of intermediary chemoattractants. End target molecules did not require chemotactic properties, since the p38 MAPK activator, LPS, also inhibited Akt and prevented migration to intermediary chemoattractants, p38 MAPK inhibitors not only reversed this hierarchy, such that neutrophils migrated preferentially toward intermediary chemoattractants, but also allowed neutrophils to be drawn out of a local end target chemoattractant environment and toward intermediary chemoattractants unexpectedly in an exaggerated (two- to fivefold) fashion. This was entirely related to significantly increased magnitude and duration of Akt activation. Finally, end target chemoattractant responses were predominantly Mac-1 dependent, whereas nondominant chemoattractants used primarily LFA-1. These data provide support for a two pathway signaling model wherein the end target chemoattractants activate p38 MAPK, which inhibits intermediary chemoattractant-induced PI3K/Akt pathway, establishing an intracellular signaling hierarchy.

Role of ICAM-1 in chromic hepatic allograft rejection in the rat. AM J Physiol Gastrointerst Liver Phsiol 283: G196-G203, 2002. First punlished February 20, 2002; 10.1152/ajpgi. 00222.2001.--the pathogenesis of hepatic allograft rejection remains unclear. We aimed to clarify the early role of intercellular adhesion molecile-1 (ICAM-1)-mediated cell recruitment in chronic hepatic injection. Liver transplanation was performed from Lewis to Lewis rats (isograft controls) and from Lewis to Brown Norway rats (allograft rejection group). The allografted rats were treated with either ICAM-1 antisense oligonucleotides (10 mg.[kg.sup-1]. [day.sup.-1] x 6 days ip) or a control preparation (either ICAM-1 missense oligonnucleotide or normal saline). Hepatic leukocyte recruitment in vivo was studied on day 6 by using intravital microscopy. Liver histology, biochemisty, and survival rates were also examined. Leukocyte adhesion in terminal hepatic vanuels was significantly increased in the rejection group compare with isograft controls. Antisense ICAM-1 in the allografted group effectively reduced leukocyte adhesion. Histology and liver chemistry were less deranged in the anisense-treated group compared with contol-treated allografted rats. In the allograft groups, survival was significantly prolonged in the antisense-treated rats (42.3 [+ or -] 1.2 days) compared with the controls (25.2 [+ or -] 2.7 days). These results showed that early leukocyte recruitment in the hepatic micorvasculature of rejection allografts is ICAM-1 dependent and suggest that impactin on early cell recruiment can signigficantly ameliorate chronic rejection. intracellular adhesion molecule 1; leukocyte; adhesion; transplantation; antisense oligonucleotide

Molecular mechanisms of leukocyte recruitment in postischemic liver microcirculation. Am J Physiol Gastrointest Liver Physiol 283: G139-G147, 2002. First published March 20, 2002; 10.1152/ajpgi.00058.2002.--Evidence shows that leukocyte recruitment into inflamed liver sinusoids does not require selectins, with one notable exception: ischemia-reperfusion (I/R). We used intravital microscopy to directly visualize the liver microcirculation during I/R and localized endotoxemia (liver superfused with lipopolysaccharide). General anti-selectin therapy (fucoidan) or anti-adhesion therapy with an antithrombin inhibitor (hirudin) was also used. Many neutrophils rolled and adhered in postsinusoidal vessels and sequestered in the sinusoids during I/R and local endotoxin superfusion. Although fucoidan blocked rolling in both forms of inflammation, leukocyte recruitment into sinusoids was only blocked in I/R. Adhesion was also inhibited in postischemic sinusoids with a second anti-adhesive agent (hirudin). Because liver I/R inevitably induces ischemia upstream in the intestine, anti-selectin therapy may prevent intestinal injury, which could prevent downstream liver inflammation. To test this hypothesis, we completely removed the intestine and rerouted blood flow from the superior mesenteric artery to the superior mesenteric vein. I/R was induced in the liver microcirculation, and many leukocytes rolled and adhered in postsinusoidal venules and adhered in sinusoids. Although fucoidan significantly reduced the rolling in postsinusoidal vessels, adhesion persisted in the sinusoids. Our data suggest that anti-adhesion therapy is effective in liver I/R in the sinusoids and postsinusoidal venules, perhaps in part due to its beneficial effect on the intestine. selectins; thrombin; adhesion; sinusoid

Author(s): Geoffrey C. Ibbotson [sup.1] , Christopher Doig [sup.1] , Jaswinder Kaur [sup.1] , Varinder Gill [sup.1] , Lena Ostrovsky [sup.1] , Todd Fairhead [sup.1] , Paul Kubes [sup.1] Author [...], Using a novel flow chamber assay system and whole blood, we show that leukocytes from septic individuals have a four-fold elevation of adhesion, but not rolling, on a P-selectin/[beta].sub.2-integrin substrate. Most leukocytes from septic patients (but not healthy controls) that bound vascular cell adhesion molecule 1 (VCAM-1) were neutrophils. All adhesion was inhibited with an antibody specific for the VCAM-1 ligand [alpha].sub.4-integrin. The [alpha].sub.4-integrin was present on neutrophils from septic patients but not on neutrophils from patients with localized bacterial infections. The plasma milieu of septic patients was sufficient to induce neutrophils from healthy subjects to bind VCAM-1 under flow conditions. This is the first description of [alpha].sub.4-integrin/VCAM-1 pathway of neutrophil recruitment in human disease. This pathway may provide a new therapeutic target to reduce inappropriate neutrophil adhesion without altering the normal yet critical [beta].sub.2-integrin-mediated adhesive function of neutrophils.

Using a novel flow chamber assay system and whole blood, we show that leukocytes from septic individuals have a four-fold elevation of adhesion, but not rolling, on a P-selectin/[beta][sub.2]-integrin substrate. Most leukocytes from septic patients (but not healthy controls) that bound vascular cell adhesion molecule 1 (VCAM-1) were neutrophils. All adhesion was inhibited with an antibody specific for the VCAM-1 ligand [alpha][sub.4]-integrin. The [alpha][sub.4]-integrin was present on neutrophils from septic patients but not on neutrophils from patients with localized bacterial infections. The plasma milieu of septic patients was sufficient to induce neutrophils from healthy subjects to bind VCAM-1 under flow conditions. This is the first description of [alpha][sub.4]-integrin/VCAM-1 pathway of neutrophil recruitment in human disease. This pathway may provide a new therapeutic target to reduce inappropriate neutrophil adhesion without altering the normal yet critical [beta][sub.2]-integrin-mediated adhesive function of neutrophils., Author(s): Geoffrey C. Ibbotson [1]; Christopher Doig [1]; Jaswinder Kaur [1]; Varinder Gill [1]; Lena Ostrovsky [1]; Todd Fairhead [1]; Paul Kubes (corresponding author) [1] Severe sepsis syndrome/septic shock is [...]

Inhaled nitric oxide (NO) was examined whether it could have an impact on the two compartments of distal organs namely the extravascular and intravascular spaces. Intravital microscopy was used to further explore the intravascular impact of inhaled NO to visualize the microvasculature. The results showed that inhaled NO could be initiated after reperfusion and still reduced microvascular disturbances including increased leukocyte adhesion and reversed impairment in blood flow.

The effect of histamine and leukotriene C4 on the leukocyte activation and adhesion in liver microcirculation was investigated. The hepatic microvasculature was visualized using intravital microscopy to assess the components and mechanisms involved in juxtacrine activation and adhesion of leukocytes. It was found that leukotriene C4 does not activate the known early mechanisms of leukocyte recruitment, including platelet-activating factor, P-selectin or intercellular adhesion molecule-1 in the hepatic microvasculature.

The role of mast cells in ischemia-reperfusion (I/R)-induced mucosal and microvascular dysfunction was investigated. I/R was induced in the intestine and skeletal muscle of wild-type mice and mast cell-deficient mice. Findings indicated that mast cells may be significant mediators of I/R-induced mucosal and microvascular dysfunction in the intestine but not fof microvascular function in skeletal muscle.

Rats sensitized to egg albumin were used in the investigation of the mesenteric venule response to mast cell activation. Intravital microscopy was used to monitor venular response, mast cell degranulation, changes in vascular permeability, adhesion of leukocytes and platelet aggregation. Inhibitory studies were also conducted to probe the mechanisms and mediators responsible for venular responses.

Intravital microscopy was used to investigate the effects of preconditioning and adenosine on neutrophil rolling, vascular dysfunction and neutrophil adhesion. Results show that while preconditioning mitigated the negative effects of ischemia and reperfusion on the dynamics of neutrophils and neutrophil-endothelial cell interactions, the effects of adenosine is minimal at best indicating that it may not have a role in the interaction between neutrophils and the endothelium.

Research shows that endogenous nitric oxide impedes the rolling interaction in leukocytes induced by tumor necrosis factor (TNF)-alpha. In an experiment designed to examine this phenomenon, cats were fasted and anesthetized using ketamine hydrochloride. Results indicate that the dose response of TNF-alpha gradually declined after showing stability in the initial stages. However, while endogenous nitric oxide reduced leukocyte rolling, exogenous nitric oxide displayed no effect.

The relationship between mast cell-induced recruitment of polymorphonuclear leukocytes (PMN) and microvascular permeability was analyzed in perivascular mast cells after exposure to compound 48/80 (CMP 48/80). Treatment of mast cells with CMP 48/80 enhanced mast cell activation, microvascular permeability and leakage of fluorescein isothiocyanate-labeled bovine albumin. Furthermore, PMN infiltration also increased 30 minutes after the administration of CMP 48/80 indicating the biphasic response of mast cell activation on microvascular permeability.

The mechanism of hydrogen peroxide (H2O2)-induced leukocyte rolling is P-selectin-dependent and the rolling response reflects regulation by endogenous antioxidant systems, including catalase, NO and glutathione. The removal of any of these three antioxidants leads to an increase in sensitivity to H2O2 which may contribute to the recruitment of leukocytes and amplification of the inflammatory response.

Exogenous administration of glucocorticoids inhibits neutrophil recruitment to inflammatory sites, but the effects of endogenously released glucocorticoids are unknown. An assessment of the effect of elevated endogenous glucocorticoid levels on leukocyte recruitment in the rat carrageenan-induced pleurisy model of acute localized inflammation shows that endogenous glucocorticoids inhibit inflammatory response by inhibiting the accumulation of leukocytes at the sites of inflammation.

The role of nitric oxide (NO) in the regulation of mucosal barrier function under physiological conditions was investigated by monitoring the effects of NO on blood flow, inhibition of platelet and leukocyte adhesion, mast cell reactivity and scavenging of reactive oxygen metabolites. The results showed that both endogenous and exogenous forms of NO apparently reduce the sequelae of acute inflammation. There is also evidence showing that NO has a role disorders such as sepsis and inflammatory bowel disease.

Monosodium urate (MSU) crystals induce neutrophil adhesion in human umbilical vein endothelial and other cells, but the adhesion mechanism is independent of CD18 and selectin. The MSU crystal-promoted neutrophil adhesion occurs by a dose- and size-dependent mechanism. Prior treatment of endothelial cells and neutrophils with colchicine lowers adhesive interaction to cellular substrata. The neutrophil adhesion is sensitive to temperature differences.

Excessive concentrations of nitric oxide in the intestine do not increase microvascular or mucosal dysfunction. Inflammatory agents do increase the levels of vascular and mucosal permeability but elevated levels of nitric oxide do not make such permeability worse or cause any cellular injury in vitro. High levels of nitric oxide may even be important therapy for various vascular diseases.

Experiments analyzing the functions of nitric oxide (NO) donors during polymorphonuclear leukocyte (PMN) activity and comparing the anti-adhesive effects of NO donors on the immunoneutralization of three adhesion glycoproteins, P-selectin, intercellular adhesion molecule-1 and CD11/CD18 reveal that NO donors significantly inhibit PMN adhesion to the postcapillary venules but not the rolling of PMNs. Anti-P-selectin antibody suppresses the flux of PMN rolling while anti-CD18 monoclonal antibody IB4 inhibits leukocyte adhesion as do NO donors. The increase in the amount of rolling PMN and the adhesion of PMN depend on P-selectin and CD18, respectively, to mediate the onset of PMN infiltration.

Ischemic reperfusion of small bowels initiates mast cell stimulation and degranulation by oxidants, leading to elevated granulocyte infiltration and mucosal dysfunction. An increase in plasma rat mast cell protease II levels, myeloperoxidase activity and epithelial permeability are caused by ischemic reperfusion. Platelet-activating factor, which is a mast cell-derived mediator, contributes to granulocyte infiltration but is not involved in epithelial permeability changes due to reperfusion.

An analysis of molecular processes related to superoxide-induced leukocyte-endothelial cell interactions in vivo reveals that the enhanced leukocyte flux, induced by superoxide, is mediated by P-selectin. Leukocyte adhesion is modulated by platelet activating factor (PAF) and adhesion molecule CD18. CD18, and not P-selectin, may be responsible for a decline in leukocyte rolling velocity, induced by PAF.

Studies to evaluate the role of mast cells and mediators from mast cells in the increased epithelial permeability, accompanying synthesis of nitric oxide in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), showed that epithelial permeability enhanced significantly due to L-NAME while mucosal permeability was reduced by two mast cell stabilizers. L-NAME-treated animals exhibited increased levels of rat mast cell protease II activity.

Nitric oxide is a homeostatic regulator of microvascular permeability in the feline small intestine, acting through specific activation of cystosolic guanylate cyclase and increased guanosine 3'5'-cyclic monophosphate (cGMP) levels. The atrial natriuretic factor causes changes in the intestinal microvascular permeability that are unrelated to cGMP levels. This is possibly due to the absence of membrane-associated guanylase cyclase in intestinal microcirculation.

A study was conducted to test whether the theory that the antiadhesive characteristics of nitric oxide (NO) are connected to superoxide production is correct by utilizing hypoxanthine-xanthine oxidase infusion to produce superoxide-dependent rolling and adhesion. It is possible for NO to act as an antiadhesion molecular in vivo, and the antiadhesion characteristics of NO are connected to its capacity to inactivate the superoxide anion.

Reperfusion of the postischemic intestine causes impairment of mucosal activity, which is ametiorated by nitric oxide synthesis-suppression. Exogenous nitric oxide sources are capable of blocking mucosal barrier destruction, as was revealed in feline intestinal ischemic studies. This is observed in SIN-1, AS-754, nitroprusside and exogenous L-arginine. Intestinal blood flow does not affect nitric oxide amelioration of mucosal dysfunction.

The role of nitric oxide (NO) in intestinal barrier function following ischemia-reperfusion was investigated. The study protocol involved intra-arterial infusion of the NO synthesis inhibitor N(super G)-nitro-L-arginine methyl ester in autoperfused cat ileum segments after ischemia-reperfusion. The results showed that NO synthesis is necessary in minimizing dysfunction of the mucosal and microvascular barrier due to intestinal ischemia-reperfusion. Inactivation of NO synthesis aggravates tissue injury.

As babies leave their mothers' wombs, they are colonized by commensal bacteria. A new study shows that these microbes in a newborn's gut can promote an increase in circulating neutrophils that can guard the body from infections. Altering a neonate's gut microbiota with perinatal antibiotic treatment can impair neutrophils, leaving the newborn vulnerable to attack by harmful bacteria (pages 524-530)., Globally, around 4 million newborn babies die yearly due to sepsis (1). Prolonged use of antibiotics is currently under clinical practice in the interest of preventing early-onset sepsis in newborns [...]

With the use of a whole blood laminar flow chamber system, we examined the types of leukocytes, adhesion molecules and the role of nuclear factor-[Kappa]B (NF-[Kappa]B) in thrombin-induced leukocyte recruitment. Primary human umbilical vein endothelial cells (HUVEC) stimulated with thrombin induced a significant increase in P-selectin-dependent neutrophil recruitment. Unexpectedly, brief thrombin stimulation (3 min) of endothelium also induced a significant lymphocyte recruitment 4 h later in addition to neutrophil recruitment. E-selectin antibody reduced neutrophil recruitment by [is greater than] 90%, whereas vascular adhesion molecule-1 (VCAM-1)[[Alpha].sub.4]-integrin were primarily responsible for lymphocyte recruitment. To examine whether NF-[Kappa]B contributed to leukocyte recruitment 4 h post thrombin stimulation, we treated HUVEC with the NF-[Kappa]B inhibitor MG-132 for 1 h before thrombin stimulation. MG-132 significantly reduced the number of rolling (77.1%) and adherent (79.9%) leukocytes compared with thrombin stimulation alone. The inhibitor was more effective at preventing lymphocyte than neutrophil recruitment, consistent with its greater effect on VCAM-1 versus E-selectin expression. Tumor necrosis factor-[Alpha]- and MG-132-treated HUVEC displayed no inhibition of leukocyte recruitment despite a decrease in NF-[Kappa]B activation. In summary, thrombin causes predominant neutrophil recruitment via rapid P-selectin expression but also a delayed E-selectin- and VCAM-1-dependent neutrophil and lymphocyte recruitment via de novo protein synthesis. Although NF-[Kappa]B mobilization was essential for thrombin-mediated VCAM-1-dependent recruitment, it only partially contributed to E-selectin-dependent recruitment. selectins; vascular adhesion molecule-1; integrin; tumor necrosis factor-[Alpha] endothelium

Previous work has demonstrated that circulating neutrophils (polymorphonuclear leukocytes [PMNs]) adhere to cardiac myocytes via [[Beta].sub.2]-integrins and cause cellular injury via the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme system. Since PMNs induced to leave the vasculature (emigrated PMNs) express the [[Alpha].sub.4]-integrin, we asked whether (a) these PMNs also induce myocyte injury via NADPH oxidase; (b) [[Beta].sub.2]-integrins (CD18) still signal oxidant production, or if this process is now coupled to the [[Alpha].sub.4]-integrin; and (c) dysfunction is superoxide dependent within the myocyte or at the myocyte--PMN interface. Emigrated PMNs exposed to cardiac myocytes quickly induced significant changes in myocyte function. Myocyte shortening was decreased by 30-50% and rates of contraction and relaxation were reduced by 30% within the first 10 min. Both [[Alpha].sub.4]-integrin antibody (Ab)-treated PMNs and NADPH oxidase--deficient PMNs were unable to reduce myocyte shortening. An increased level of oxidative stress was detected in myocytes within 5 min of PMN adhesion. Addition of an anti-[[Alpha].sub.4]-integrin Ab, but not an anti-CD18 Ab, prevented oxidant production, suggesting that in emigrated PMNs the NADPH oxidase system is uncoupled from CD18 and can be activated via the [[Alpha].sub.4]-integrin. Addition of exogenous superoxide dismutase (SOD) inhibited all parameters of dysfunction measured, whereas overexpression of intracellular SOD within the myocytes did not inhibit the oxidative stress or the myocyte dysfunction caused by the emigrated PMNs. These findings demonstrate that profound molecular changes occur within PMNs as they emigrate, such that CD18 and associated intracellular signaling pathways leading to oxidant production are uncoupled and newly expressed [[Alpha].sub.4]-integrin functions as the ligand that signals oxidant production. The results also provide pathological relevance as the emigrated PMNs have the capacity to injure cardiac myocytes through the [[Alpha].sub.4]-integrin--coupled NADPH oxidase pathway that can be inhibited by extracellular, but not intracellular SOD. Key words: heart * neutrophils * integrins * free radicals * oxidant

Thrombin and leukocyte recruitment in endotoxemia. Am J Physiol Heart Circ Physiol 279: H1338-H1345, 2000.--Because thrombin has been implicated in sepsis, it has been proposed that antithrombin III (AT III) is beneficial due to its anticoagulatory and antiadhesive effects. Using intravital microscopy, we visualized leukocyte-endothelium interactions in postcapillary venules of the reline mesentery exposed to lipopolysaccharide (LPS). At a concentration of AT III that blocks leukocyte adhesion in postischemic mesentery, we found no role for thrombin in LPS-induced rolling, adhesion and emigration, or microvascular dysfunction. Furthermore, AT III did not attenuate leukocyte-endothelial interactions after tumor necrosis factor-[Alpha] superfusion of the mesentery. In contrast, fucoidan, a selectin inhibitor, prevented almost all LPS-induced rolling and reduced adhesion, emigration, and microvascular dysfunction. In a model of endotoxemia, leukocyte recruitment into mesentery or lungs was unaffected by AT III. Finally, in a human cell system that mimics the flow conditions in vivo, human neutrophils rolled, adhered, and emigrated similar to the feline postcapillary microvessels, and AT III had no effect on leukocyte recruitment induced by LPS. If AT III has beneficial effects in endotoxemia, it is not due to a direct effect upon leukocyte rolling, adhesion, or emigration in postcapillary venules in vivo. sepsis; endotoxin; adhesion; antithrombin III

Spontaneously developing chronic colitis in IL-10/iNOS double-deficient mice. Am J Physiol Gastrointest Liver Physiol 279: G90-G99, 2000.--Mice deficient in both inducible nitric oxide synthase (iNOS) and interleukin (IL)-10 ([iNOS.sup.-/-]/IL-[10.sup.-/-]) were created to examine the role of iNOS in spontaneously developing intestinal inflammation. IL-[10.sup.-/-]/[iNOS.sup.-/-] mice were compared with IL-[10.sup.-/-] ([iNOS.sup.+/+]) littermates over 6 mo. RT-PCR, Western blot analysis, and immunohistochemistry were performed to measure iNOS message and protein levels. Plasma nitrate/nitrite ([NO.sub.x]) levels were assessed by HPLC. Damage scores (macroscopic and microscopic) and granulocyte infiltration were assessed. At 3-4 wk, IL-[10.sup.-/-] and IL-[10.sup.-/-]/[iNOS.sup.-/-] mice had no signs of colonic inflammation or granulocyte infiltration. Plasma [NO.sub.x] levels were not different from controls. By 3-4 mo, IL-[10.sup.-/-] mice had increased damage scores and granulocyte infiltration concurrent with increased mRNA and protein synthesis (restricted to the epithelium) for iNOS in intestinal tissues but not other tissues. Plasma [NO.sub.x] levels increased fivefold. Interestingly, in the absence of iNOS induction or increased plasma [NO.sub.x], [iNOS.sup.-/-]/IL-[10.sup.-/-] mice had damage and granulocyte infiltration equivalent to those observed in IL-[10.sup.-/-] littermates. These data suggest that iNOS does not impact on the development or severity of spontaneous chronic inflammation in IL-10-deficient mice. inflammatory bowel disease; nitric oxide; intestine; inflammation; myeloperoxidase

Ostrovsky, Lena, Juliana Carvalho-Tavares, Richard C. Woodman, and Paul Kubes. Translational inhibition of E-selectin expression stimulates P-selectin-dependent neutrophil recruitment. Am J Physiol Heart Circ Physiol 278: H1225-H1232, 2000.--Although known for its role in hemostasis, there is a growing body of evidence that thrombin can induce leukocyte recruitment and contribute to the inflammatory response. An in vitro parallel-plate flow chamber was used to systematically examine thrombin-induced neutrophil interactions with human endothelium. Stimulation of endothelial cells with thrombin (1 U/ml) resulted in an immediate, P-selectin-dependent increase in neutrophil rolling and adhesion that was comparable in magnitude to optimal levels of histamine (the classical inducer of P-selectin). However, thrombin, but not histamine, induced a delayed (4 h) E-selectin-dependent rolling similar to that of tumor necrosis factor-[Alpha], suggesting that thrombin has the unique ability to recruit neutrophils by an early P-selectin and a delayed E-selectin pathway. Surprisingly, inhibition of E-selectin expression with the general protein synthesis inhibitor cycloheximide induced P-selectin expression 4 h after thrombin stimulation. Cycloheximide and thrombin (4 h) induced sufficient P-selectin-dependent rolling to recruit as many neutrophils as were recruited with 4 h of stimulation with thrombin alone. Histamine in the presence of cycloheximide or cycloheximide alone did not evoke the P-selectin response at 4 h, suggesting that this was not due to direct cycloheximide induction of P-selectin. Treatment of endothelium with tumor necrosis factor-[Alpha] (an E-selectin inducer) and cycloheximide also eliminated E-selectin expression but, much like thrombin, induced P-selectin expression and neutrophil recruitment. In conclusion, inhibition of E-selectin via protein synthesis inhibition activates the protein synthesis-independent pathway of P-selectin expression to support adequate leukocyte recruitment. leukocytes; endothelium; adhesion molecules; thrombin; tumor necrosis factor