Priming Of Polymorphonuclear Neutrophils Research Articles

Periodontal Inflammation Primes the Systemic Innate Immune Response

The presence of periodontal diseases (PDs) often strongly correlates with other severe chronic inflammatory conditions, including cardiovascular disease, diabetes, and arthritis. However, the mechanisms through which these diseases interact are unclear. In PD, tissue and bone destruction in the mouth is driven by elevated recruitment of polymorphonuclear neutrophils (PMNs), which are primed and recruited from the circulation to sites of inflammation. We predicted that systemic effects on PMN mobilization or priming could account for the interaction between PD and other inflammatory conditions. We tested this using a mouse model of ligature-induced PD and found elevated PMN counts specifically in bone marrow, supporting a systemic effect of periodontal tissue inflammation on PMN production. In contrast, mice with induced peritonitis had elevated PMN counts in the blood, peritoneum, and colon. These elevated counts were further significantly increased when acute peritonitis was induced after ligature-induced PD in mice, revealing a synergistic effect of multiple inflammatory events on PMN levels. Flow cytometric analysis of CD marker expression revealed enhanced priming of PMNs from mice with both PD and peritonitis compared to mice with peritonitis alone. Thus, systemic factors associated with PD produce hyperinflammatory PMN responses during a secondary infection. To analyze this systemic effect in humans, we induced gingival inflammation in volunteers and also found significantly increased activation of blood PMNs in response to ex vivo stimulation, which reverted to normal following resolution of gingivitis. Together, these results demonstrate that periodontal tissue inflammation has systemic effects that predispose toward an exacerbated innate immune response. This indicates that peripheral PMNs can respond synergistically to simultaneous and remote inflammatory triggers and therefore contribute to the interaction between PD and other inflammatory conditions. This suggests larger implications of PD beyond oral health and reveals potential new approaches for treating systemic inflammatory diseases that interact with PD.

Characterization of neutrophil-neuronal co-cultures to investigate mechanisms of post-ischemic immune-mediated neurotoxicity

BackgroundImmune-mediated reperfusion injury is a critical component of post-ischemic central nervous system (CNS) damage. In this context, the activation and recruitment of polymorphonuclear neutrophils (PMNs) to the CNS induces neurotoxicity in part through the release of degradative enzymes, cytokines, and reactive oxygen species. However, the extent to which close-range interactions between PMNs and neurons contribute to injury in this context has not been directly investigated. New methodWe devised a co-culture model to investigate mechanisms of PMN-dependent neurotoxicity. Specifically, we established the effect of PMN dose, co-incident neuronal ischemia, lipopolysaccharide (LPS)-induced PMN priming, and the requirement for cell-cell contact on cumulative neuron damage. Results and comparison to existing method(s)Pre-exposure of day in vitro 10 primary cortical neurons to oxygen-glucose deprivation (OGD) enhanced PMN-dependent neuronal death. Likewise, LPS-induced priming of the PMN donor further increased PMN-induced toxicity in vitro compared to saline-injected controls. Compartmentalization of LPS-primed PMNs using net wells confirmed the requirement for close-range cell-cell interactions in the process of PMN-induced neuronal injury. Moreover, time-lapse imaging and quantitative neurite analyses implicate PMN-neurite interactions in this pathological response. These experiments establish a platform to investigate immune and neural factors that contribute to post-ischemic neurodegeneration. ConclusionsIschemic and immune priming enhance neurotoxicity in PMN-neuronal co-cultures. Moreover, cell-cell contact and neurite destruction are prominent features in the observed mechanism of post-ischemic neuronal death.

Extracellular Chromatin Triggers Release of Soluble CEACAM8 Upon Activation of Neutrophils.

Increased concentrations of extracellular chromatin are observed in cancer, sepsis, and inflammatory autoimmune diseases like systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). In SLE and RA, extracellular chromatin may behave as a danger-associated molecular pattern (DAMP). Polymorphonuclear neutrophils (PMN) are described as typical pro-inflammatory cells but possess also immunoregulatory properties. They are activated in SLE and RA but surprisingly remain moderately studied in these diseases, and especially the disease-associated stimuli triggering PMN activation are still not completely characterized. PMN express plasma membrane carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 8 (CD66b) and secrete a soluble form of CEACAM8 after activation. Soluble CEACAM8 has in turn immunoregulatory functions. However, few natural stimuli inducing soluble CEACAM8 secretion by PMN have been identified. Here we demonstrate for the first time that extracellular chromatin triggers secretion of soluble CEACAM8 by primary human PMN. Priming of PMN was not required. Secretion was associated with activation of PMN. Similar induction of soluble CEACAM8 release was observed with purified mono-nucleosomes as well as long chromatin fragments and occurred in a time-dependent and concentration-dependent manner. Results indicate that chromatin induces both neo-synthesis of soluble CEACAM8 and release of soluble CEACAM8 through degranulation. In addition, we report the presence of soluble CEACAM8 at high concentration in the synovial fluid of RA patients. Thus, we describe here a novel mechanism by which a natural DAMP, with inflammatory properties in SLE and RA, induces soluble CEACAM8 secretion by activated PMN with potential immunoregulatory consequences on other immune cells, including PMN.

Impact of CD14 on Reactive Oxygen Species Production from Human Leukocytes Primed by Escherichia coli Lipopolysaccharides.

Lipopolysaccharides (LPS) from Gram-negative bacteria prime human polymorphonuclear neutrophils (PMNs) via multicomponent receptor cluster including CD14 and MD-2·TLR4 for the enhanced release of reactive oxygen species (ROS) were triggered by bacterial derived peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). In this study, we investigated the impact of CD14 on LPS-induced priming of human PMNs for fMLP-triggered ROS generation (respiratory or oxidative) burst. Monoclonal antibodies against human CD14 (mAbs) as well as isotype-matched IgG2a did not influence significantly fMLP-triggered ROS production from LPS-unprimed PMNs. Anti-CD14 mAbs (clone UCHM-1) attenuated LPS-induced priming of PMNs as it had been mirrored by fMLP-triggered decrease of ROS production. Similar priming activity of S-LPS or Re-LPS from Escherichia coli for fMLP-triggered ROS release from PMNs was found. Obtained results suggest that glycosylphosphatidylinositol-anchored CD14 is the key player in LPS-induced PMN priming for fMLP-triggered ROS production. We believe that blockade of CD14 on the cell surface and clinical use of anti-CD14 mAbs or their Fab fragments may diminish the production of ROS and improve outcomes during cardiovascular diseases manifested by LPS-induced inflammation.

Abstract WP351: Lung-Specific Extracellular Superoxide Dismutase Blocks Neutrophil-Mediated Toxicity in Brain Ischemia-Reperfusion

Introduction: Tissue damage and neuroinflammatory responses associated with cerebral ischemia trigger a systemic immune response that exacerbates brain injury during reperfusion and adversely influences functional recovery. Polymorphonuclear neutrophils (PMNs) are the first-line response to inflammation following acute brain injury, and evidence implicates lung-dependent PMN depriming as a critical endogenous protective mechanism against reperfusion injury. Methods: We explored mechanisms of PMN priming and neurotoxicity with in vivo modeling of systemic inflammation after brain ischemia-reperfusion as well as cellular culture experiments. Using transgenic mice overexpressing the human antioxidant gene superoxide dismutase 3 in the lung (TgSOD3), we tested the hypothesis that manipulation of the lung’s redox environment could mitigate PMN activation and post-ischemic brain injury. Injury was induced by occlusion of the common carotid and basilar arteries followed by reperfusion and lipopolysaccharide-induced systemic inflammation. To assess cell interactions using live microscopy, we cultured primary cortical neurons after oxygen-glucose deprivation (OGD) with primed PMNs from wild-type or TgSOD3 mice. Results: Our results show that pulmonary SOD3 protects against peripheral PMN activation, lung injury, brain injury, and neuroinflammation after ischemia/reperfusion with systemic inflammation. We also found that PMNs from TgSOD3 mice are less toxic to post-OGD cultures compared to wild-type PMNs. Our data indicate that direct cell-cell contact is required for PMN-dependent neurotoxicity in mixed cellular cultures and that pre-exposure of neurons to OGD amplifies this response. Analyses reveal that PMNs effect a considerable degree of neuritic damage in vitro , while in vivo modeling demonstrates cortical injury, blood-brain barrier compromise, PMN activation, and lung injury after reperfusion. Conclusion: Collectively, these studies expand our understanding of PMN-dependent mechanisms in post-ischemic toxicity, both directly on neurons and via effects on CNS inflammation, and argue that the inflammatory milieu within the lung may modulate CNS reperfusion injury via manipulation of peripheral PMN priming.

Post-Traumatic Acute Lung Injury: The Role of Succinate

Acute lung injury (ALI) occurs in 44% of patients after hemorrhagic shock (HS) and carries a high morbidity. Polymorphonuclear neutrophils (PMNs) are central to the pathogenesis of post-traumatic ALI. PMNs become primed, with an enhanced response to stimuli after injury and subsequently can induce ALI after a second insult. Succinate is a metabolite that accumulates after injury (up to 700uM from physiologic of 1uM) and has pro-inflammatory effects. We hypothesize that succinate is a causative factor in ALI through PMN priming.

Prokaryotic RNA Associated to Bacterial Viability Induces Polymorphonuclear Neutrophil Activation.

Polymorphonuclear neutrophils (PMN) are the first cellular line of antibacterial host defense. They sense pathogens through recognition of pathogen-associated molecular patterns (PAMPs) by innate pattern recognition receptors, such as Toll-like receptors (TLR). The aim of this study was to investigate whether PMN sense bacterial viability and explore which viability factor could be involved in this phenomenon. For this purpose, different functions were evaluated in isolated human PMN using live Escherichia coli (Ec) and heat-killed Ec (HK-Ec). We found that bacterial viability was indispensable to induce PMN activation, as measured by forward-scatter (FSC) increase, CD11b surface expression, chemotaxis, reactive oxygen species (ROS) generation and neutrophil extracellular trap (NET) formation. As uncapped non-polyadenylated prokaryotic mRNA has been recognized as a PAMP associated to bacterial viability by macrophages and dendritic cells, total prokaryotic RNA (pRNA) from live Ec was purified and used as a stimulus for PMN. pRNA triggered similar responses to those observed with live bacteria. No RNA could be isolated from HK-Ec, explaining the lack of effect of dead bacteria. Moreover, the supernatant of dead bacteria was able to induce PMN activation, and this was associated with the presence of pRNA in this supernatant, which is released in the killing process. The induction of bactericidal functions (ROS and NETosis) by pRNA were abolished when the supernatant of dead bacteria or isolated pRNA were treated with RNAse. Moreover, endocytosis was necessary for pRNA-induced ROS generation and NETosis, and priming was required for the induction of pRNA-induced ROS in whole blood. However, responses related to movement and degranulation (FSC increase, CD11b up-regulation, and chemotaxis) were still triggered when pRNA was digested with RNase, and were not dependent on pRNA endocytosis or PMN priming. In conclusion, our results indicate that PMN sense live bacteria through recognition of pRNA, and this sensing triggers potent bactericidal mechanisms.

Tolerance to lipopolysaccharide promotes an enhanced neutrophil extracellular traps formation leading to a more efficient bacterial clearance in mice

Tolerance to lipopolysaccharide (LPS) constitutes a stress adaptation, in which a primary contact with LPS results in a minimal response when a second exposure with the same stimulus occurs. However, active important defence mechanisms are mounted during the tolerant state. Our aim was to assess the contribution of polymorphonuclear neutrophils (PMN) in the clearance of bacterial infection in a mouse model of tolerance to LPS. After tolerance was developed, we investigated in vivo different mechanisms of bacterial clearance. The elimination of a locally induced polymicrobial challenge was more efficient in tolerant mice both in the presence or absence of local macrophages. This was related to a higher number of PMN migrating to the infectious site as a result of an increased number of PMN from the marginal pool with higher chemotactic capacity, not because of differences in their phagocytic activity or reactive species production. In vivo, neutrophils extracellular trap (NET) destruction by nuclease treatment abolished the observed increased clearance in tolerant but not in control mice. In line with this finding, in vitro NETs formation was higher in PMN from tolerant animals. These results indicate that the higher chemotactic response from an increased PMN marginal pool and the NETs enhanced forming capacity are the main mechanisms mediating bacterial clearance in tolerant mice. To sum up, far from being a lack of response, tolerance to LPS causes PMN priming effects which favour distant and local anti-infectious responses.

Effects of interferon-γ and granulocyte colony-stimulating factor on antifungal activity of human polymorphonuclear neutrophils against Candida albicans grown as biofilms or planktonic cells

Candida albicans is a leading cause of biofilm-related infections. As Candida biofilms are recalcitrant to host defenses, we sought to determine the effects of interferon-γ and granulocyte colony-stimulating factor, two pro-inflammatory cytokines, on the antifungal activities of human polymorphonuclear neutrophils (PMNs) against C. albicans biofilms, using an in vitro biofilm model. Priming of PMNs by these cytokines augmented fungal damage of planktonic cells; however, priming of PMNs did not have the same effect against Candida biofilms. Biofilm phenotype appears to play an important role in protecting C. albicans from the innate immune system.

Amantadine inhibits platelet-activating factor induced clathrin-mediated endocytosis in human neutrophils.

Receptor signaling is integral for adhesion, emigration, phagocytosis, and reactive oxygen species production in polymorphonuclear neutrophils (PMNs). Priming is an important part of PMN emigration, but it can also lead to PMN-mediated organ injury in the host. Platelet-activating factor (PAF) primes PMNs through activation of a specific G protein-coupled receptor. We hypothesize that PAF priming of PMNs requires clathrin-mediated endocytosis (CME) of the PAF receptor (PAFr), and, therefore, amantadine, known to inhibit CME, significantly antagonizes PAF signaling. PMNs were isolated by standard techniques to >98% purity and tested for viability. Amantadine (1 mM) significantly inhibited the PAF-mediated changes in the cellular distribution of clathrin and the physical colocalization [fluorescence resonance energy transfer positive (FRET+)] of early endosome antigen-1 and Rab5a, known components of CME and similar to hypertonic saline, a known inhibitor of CME. Furthermore, amantadine had no effect on the PAF-induced cytosolic calcium flux; however, phosphorylation of p38 MAPK was significantly decreased. Amantadine inhibited PAF-mediated changes in PMN physiology, including priming of the NADPH oxidase and shape change with lesser inhibition of increases in CD11b surface expression and elastase release. Furthermore, rimantadine, an amantadine analog, was a more potent inhibitor of PAF priming of the N-formyl-methionyl-leucyl-phenylalanine-activated oxidase. PAF priming of PMNs requires clathrin-mediated endocytosis that is inhibited when PMNs are pretreated with either amantadine or rimantadine. Thus, amantadine and rimantadine have the potential to ameliorate PMN-mediated tissue damage in humans.

Effects of L-citrulline oral supplementation on polymorphonuclear neutrophils oxidative burst and nitric oxide production after exercise

Seventeen volunteer male professional cyclists were randomly assigned to control or supplemented (6 g L-citrulline-malate) groups and participated in a cycling stage. Blood samples were taken in basal conditions, after the race and 3 h post-race. Citrulline supplementation significantly increased plasma concentration of both arginine and citrulline after the stage only in the supplemented group. Polymorphonuclear neutrophils (PMNs) from controls responded to exercise with a progressive decrease in ROS production. Supplemented PMNs significantly increased ROS production after exercise compared to basal values and diminished to values lower than basal at recovery. PMN nitrite concentration was significantly higher after exercise and recovery only in the supplemented group. Markers of oxidative damage—CK, LDH, malondialdehyde—and DNA damage remained unchanged in both groups. In conclusion, oral L-citrulline administration previous to a cycling stage increases plasma arginine availability for NO synthesis and PMNs priming for oxidative burst without oxidative damage.

Regulation of human polymorphonuclear leukocytes functions by the neuropeptide pituitary adenylate cyclase-activating polypeptide after activation of MAPKs.

Anti-inflammatory activities of pituitary adenylate cyclase-activating protein (PACAP) are mediated in part through specific effects on lymphocytes and macrophages. This study shows that in human polymorphonuclear neutrophils (PMNs), PACAP acts as a proinflammatory molecule. In PMNs, vaso-intestinal peptide/PACAP receptor 1 (VPAC-1) was the only receptor found to be expressed by RT-PCR. Using VPAC-1 Ab, we found that VPAC-1 mRNA was translated into proteins. In PMNs, PACAP increases cAMP, inositol triphosphate metabolites, and calcium. It activates two of the three members of the MAPK superfamily, the ERK and the stress-activated MAPK p38. U73122, an inhibitor of phospholipase C (PLC), inhibits PACAP-induced ERK activation, whereas p38 MAPK phosphorylation was unaffected. Using specific pharmalogical inhibitors of ERK (PD098059) and p38 MAPK (SB203580), we found that PACAP-mediated calcium increase was ERK and PLC dependent and p38 independent. PACAP primes fMLP-associated calcium increase; it also primes fMLP activation of the respiratory burst as well as elastase release, these last two processes being ERK and PLC dependent and p38 MAPK independent. PACAP also increases membrane expression of CD11b and release of lactoferrin and metallo proteinase-9 (MMP-9). These effects were PLC dependent (CD 11b, lactoferrin, MMP-9), ERK dependent (CD 11b, lactoferrin, MMP-9), and p38 dependent (CD11b, lactoferrin). We conclude that PACAP is a direct PMN activator as well as an effective PMN priming agent that requires PLC, ERK, and p38 MAPK activities.

AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target for N-acetylcysteine treatment in dialysis patients

AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target forN-acetylcysteine treatment in dialysis patients. Oxidative stress largely contributes to hemodialysis-associated lethal complications, thus explaining the urgent need of antioxidant-based therapeutic strategies in hemodialysis patients. We previously identified advanced oxidation protein products (AOPP) in the uremic plasma as exquisite markers of oxidative stress and potent mediators of monocyte activation. The present study was aimed at searching whether (1) AOPP can also trigger activation of polymorphonuclear neutrophils (PMN), and (2) whether AOPP-induced activation could be inhibited by N-acetylcysteine (NAC), a widely used compound which has been shown to prevent oxidative injury to kidney. Both human serum albumin (HAS) AOPP (i.e., HOCl-modified HSA in vitro preparations and AOPP extracted from plasma of hemodialysis patients) were tested for their capacity to trigger phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent activities as measured by lucigenin- and luminol-amplified chemiluminescence (CL), respectively, as compared to receptor-dependent [opsonized zymosan or receptor-independent phorbol myristate acetate (PMA)]. The effect of PMN priming by platelet-activating factor (PAF), and the effect of NAC on normal monocyte and on normal or hemodialysis patient's (N = 16) PMN oxidative responses were compared. HSA-AOPP triggered in a HOCl dose-dependent manner both NADPH-oxidase- and MPO-dependent CL of PMN. This latter was further enhanced by PAF priming. Plasma-derived AOPP obtained from hemodialysis patients also triggered PMN respiratory burst. NAC significantly reduced HSA-AOPP-mediated responses of normal monocyte and of normal and uremic PMN but had no significant effect on opsonized zymosan- or PMA-induced CL responses. This dual potential of NAC to inhibit phagocyte oxidative responses induced by HSA-AOPP without affecting those mediated by compounds mimicking pathogens supports the proposal of a therapeutic trial with NAC aimed at reducing oxidative stress-related inflammation in hemodialysis patients.

Shock Mesenteric Lymph-Induced Rat Polymorphonuclear Neutrophil Activation and Endothelial Cell Injury Is Mediated by Aqueous Factors

After trauma and hemorrhagic shock (T/HS), mesenteric lymph (ML) activates polymorphonuclear neutrophils (PMNs), injures endothelial cells (ECs), and predisposes to lung injury. The involved mediators, however, are unknown. We studied the ability of aqueous (AQ) and lipid (LIP) extracts of rat T/HS ML to activate PMNs and injure ECs. ML was collected from male rats undergoing trauma (laparotomy) plus hemorrhagic shock (30 mm Hg, 90 minutes) or sham shock. AQ and LIP ML fractions were separated using the Bligh-Dyer technique. Human umbilical vein endothelial cells were incubated 18 hours in 5% LIP or AQ lymph fractions and viability was assessed using the MTT assay. Rat PMNs incubated 5 minutes with 3% LIP or AQ fractions were assessed for respiratory burst (RB) and cytosolic calcium ([Ca(2+)](i)) using dihydrorhodamine 123 and fura-2AM. Human PMN responses to AQ and LIP T/HS lymph were studied similarly. EC incubated in AQ showed 19 +/- 4% viability as compared with 65 +/- 11% in LIP (p < 0.001). Whole lymph affected ECs comparably to AQ T/HS lymph. Rat PMN basal [Ca(2+)](i) increased after exposure to AQ but not LIP T/HS lymph extracts. AQ T/HS lymph primed [Ca(2+)](i) responses to macrophage inflammatory protein-2 and platelet-activating factor; neither LIP T/HS nor any trauma and sham shock lymph fraction caused PMN priming. Rat PMN RB was elevated after AQ T/HS lymph incubation when compared with buffer (610 +/- 122 U/s vs. 225 +/- 38 U/s, p = 0.01). Rat PMN incubation in LIP T/HS lymph caused minimal activation (289 +/- 28 U/s, p = NS). Conversely, human PMN showed [Ca(2+)](i) and RB priming by rat T/HS LIP and not AQ extracts. T/HS mesenteric lymph contains multiple biologically active mediators. Both AQ and LIP extracts of T/HS lymph are toxic to human umbilical vein endothelial cells, with AQ more active than LIP. Only AQ T/HS lymph activates rat PMNs, although LIP rat lymph extract activates human PMNs. These findings demonstrate the complex nature of gut lymph-derived biologic factors as well as species-specific differences on PMN and EC physiology. Therapies directed at any one specific molecule or mediator are therefore unlikely to be successful.

Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition.

Although proteinase 3 (PR3) is known to have the potential to promote inflammation and injure tissues, the biologic forms and function of PR3 in polymorphonuclear neutrophils (PMN) from healthy donors have received little attention. In this paper, we show that PMN contain 3.24 +/- SD 0.24 pg of PR3 per cell, and that the mean concentration of PR3 in azurophil granules of PMN is 13.4 mM. Low levels of PR3 are detectable on the cell surface of unstimulated PMN. Exposure of PMN to cytokines or chemoattractants alone induces modest (1.5- to 2.5-fold) increases in cell surface-bound PR3. In contrast, brief priming of PMN with cytokines, followed by activation with a chemoattractant, induces rapid and persistent, 5- to 6-fold increases in cell surface expression of PR3, while causing minimal free release of PR3. Membrane-bound PR3 on PMN is catalytically active against Boc-Alanine-Alanine-Norvaline-thiobenzyl ester and fibronectin, but in marked contrast to soluble PR3, membrane-bound PR3 is resistant to inhibition by physiologic proteinase inhibitors. PR3 appears to bind to the cell surface of PMN via a charge-dependent mechanism because exposure of fixed, activated PMN to solutions having increasing ionic strength results in elution of PR3, HLE, and CG, and there is a direct relationship between their order of elution and their isoelectric points. These data indicate that rapidly inducible PR3 expressed on the cell surface of PMN is an important bioactive form of the proteinase. If PR3 expression on the cell surface of PMN is dysregulated, it is well equipped to amplify tissue injury directly, and also indirectly via the generation of autoantibodies.

The immunoregulatory abilities of polymorphonuclear neutrophils in the course of multiple sclerosis.

The polymorphonuclear neutrophils (PMN) possess sufficient potential to affect both immune response and inflammation, however it has not been yet described in the course of multiple sclerosis (MS). We have studied binding of fluorescein isothiocyanate (FITC)- stained TNF-alpha by PMN, the expression of CD11a, CD11b, and CD18 molecules of beta2-integrines and the expression of CD10 (neutral endopeptidase-NEP) and of CD13 (aminopeptidase N; APN) antigens on PMN in three different groups of MS patients. The control group included neurological patients (OND) with noninflammatory diseases. The obtained results have proved that during MS exacerbation and in the course of chronic progressive MS, PMN reveal several forms of preactivation, including significantly higher stained-TNF-alpha binding, higher expression of CD11b and CD18, as well as CD10 and CD13 antigens, in comparison with MS remission or OND. We suggest that the increased expression of these molecules on PMN of MS patients in exacerbation of the disease and to a lower degree in the course of CP-MS is a result of PMN priming, and directly prove the PMN involvement in the disease pathogenesis.

Maintenance and down-regulation of primed neutrophil chemiluminescence activity in human whole blood.

Priming of polymorphonuclear neutrophils (PMN) in whole blood (by tumor necrosis factor alpha and interleukin-8 for enhancement of luminol-dependent chemiluminescence induced by human complement-opsonized zymosan) was stable for 120 min. In contrast, priming of isolated PMN in plasma-free suspension for responses to opsonized zymosan, formyl-methionyl-leucyl-phenylalanine, and phorbol myristate acetate was markedly less stable. Decay of priming was not due to irreversible inactivation of the terminal CL production machinery because PMN could be reprimed by platelet-activating factor or leukotriene B4. The tumor necrosis factor-alpha-primed state of isolated PMN was stabilized by host plasma in a concentration-dependent fashion. We conclude that PMN priming results in a dynamic state that is reversible. Our findings suggest the existence of blood-borne components that may act to stabilize or modify PMN priming.

Early neutrophil sequestration after injury: a pathogenic mechanism for multiple organ failure.

Polymorphonuclear neutrophils (PMNs) play a pivotal role in the inflammation that precedes multiple organ failure (MOF). In a rat model of MOF, PMNs become primed for enhanced superoxide anion (O2-) release and CD11b expression, sequester in end organs, and produce organ failure. Therefore, we hypothesized that circulating PMNs harvested in the first 24 hours after injury from trauma patients at risk for MOF would (1) exhibit a primed O2- release, (2) upregulate CD11b expression, and (3) show evidence of sequestration in tissues. Extracellular PMN O2- release and CD11b receptor expression were measured at 3, 6, 12, and 24 hours after injury in 33 torso trauma patients with Injury Severity Scores > 15; eight patients (24%) developed MOF. Healthy adults served as controls. PMNs after injury were primed for enhanced in vitro O2- release at 3, 6, 12, and 24 hours after injury, indicating prior in vivo priming. CD11b expression was also increased at 6, 12, and 24 hours after injury. Circulating PMN numbers increased sharply at 3 hours after injury, before decreasing dramatically at 6 and 12 hours, suggesting end organ sequestration. At 12 hours after injury, declines in circulating PMNs were significantly greater in MOF than in non-MOF patients (p < 0.05). These data indicate that PMNs are quickly mobilized into the circulation after injury and then primed for enhanced O2- release and CD11b expression. PMN priming appears to be a necessary preamble to PMN sequestration in patients with major torso trauma. Upregulation of PMN function, accompanied by subsequent end organ sequestration, may represent an important early event in the pathogenesis of MOF after injury.

Postinjury neutrophil priming and activation: an early vulnerable window.

Generation of extracellular, cytotoxic superoxide anion (O2-) by polymorphonuclear neutrophils (PMNs) contributes to an unbridled inflammatory response that can precipitate multiple organ failure (MOF). Release of O2- is markedly enhanced when activated PMNs have been previously "primed" by inflammatory mediators, such as those expressed after trauma. We therefore hypothesized that PMN priming occurs as an integral part of the early inflammatory response to trauma. PMNs were obtained from 17 high-risk patients with torso trauma at 3, 6, 12, 24, 48, and 72 hours after injury, as well as from 10 healthy donors, and the in vitro release of O2- was quantitated with a kinetic, superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay. PMN O2- release was measured in the presence and absence of 1 mumol/L N-formyl-methionyl-leucyl-phenylalanine (fMLP) and after priming and activation with 20 nmol/L platelet-activating factor (PAF) and 1 mumol/L fMLP, respectively. In vitro PMN O2- release was used to determine whether postinjury PMNs were (1) activated in vivo, (2) primed in vivo, or (3) primable in vitro. Unstimulated PMNs from trauma patients spontaneously expressed modest amounts of O2- in vitro from 6 to 48 hours after injury, suggesting endogenous activation. Also, fMLP-activated PMNs collected between 3 and 24 hours after injury expressed more O2- than controls (p < or = 0.02), indicating in vivo, trauma-related priming. Furthermore, postinjury PMNs were maximally primed in vivo (i.e., in vitro exposure to PAF before fMLP activation failed to significantly enhance O2- release) as compared to PMNs treated with fMLP. These data indicate that major torso trauma (first hit) primes and activates PMNs within 3 to 6 hours after injury. Consequently, we postulate that postinjury priming of PMNs may create an early vulnerable window during which a second hit (e.g., a secondary operation or delayed hemorrhage) activates exuberant PMN O2- release, rendering the injured patient at high risk for MOF.

CD11b Blockade Prevents Lung Injury despite Neutrophil Priming after Gut Ischemia/Reperfusion

Gut ischemia/reperfusion (I/R) provokes lung injury via a mechanism that involves neutrophils [polymorphonuclear neutrophils (PMNs)]. CD11b/CD18 (alpha mB2) is the integrin receptor on PMNs critical for adhesion-dependent oxidative burst. The purpose of this study was to investigate the mechanistic role of CD11b in the process of gut I/R-induced lung injury. Sprague-Dawley rats underwent 45 minutes of superior mesenteric artery (SMA) occlusion with and without CD11b monoclonal antibody treatment (IB6) (1 mg/kg, i.v.), before SMA clamping. At 2-hour reperfusion, PMN presence in tissue was quantitated by myeloperoxidase activity and circulating PMN priming determined by the difference in superoxide production with and without N-formyl-methionyl-leucyl-phenylalanine, whereas lung leak was assessed by 125I-albumin lung/blood ratio. In sum, CD11b blockade prevented gut I/R-induced lung leak, but did not attenuate gut I/R-induced PMN priming or tissue PMN accumulation. In conclusion, gut I/R promotes PMN priming and PMN adhesion in both local and distant beds via receptors other than CD11b, but this B2 integrin receptor is critical for PMN-mediated endothelial injury.