Neutrophil L-selectin Expression Research Articles

Sialic Acid Conjugate-Modified Liposomal Dexamethasone Palmitate Targeting Neutrophils for Rheumatoid Arthritis Therapy: Influence of Particle Size.

Many anti-inflammatory therapies targeting neutrophils have been developed so far. A sialic acid (SA)-modified liposomal (SAL) formulation, based on the high expression of L-selectin in peripheral blood neutrophils (PBNs) and SA as its targeting ligand, has proved to be an effective neutrophil-mediated drug delivery system targeting rheumatoid arthritis (RA). The objective of this study was to investigate the influence of particle size of drug-carrying SALs transported and delivered by neutrophils on their anti-RA effect. Dexamethasone palmitate-loaded SALs (DP-SALs) of different particle sizes (300.2 ± 5.5nm, 150.3 ± 4.3nm, and 75.0 ± 3.9nm) were prepared with DP as a model drug. Our study indicated that DP-SALs could efficiently target PBNs, with larger liposomes leading to higher drug accumulation in cells. However, a high intake of large DP-SALs by PBNs inhibited their migration ability and capacity to release the payload at the target site. In contrast, small DP-SALs (75.0 ± 3.9nm) could maintain the drug delivery potential of PBNs, leading to their efficient accumulation at the inflammatory site, where PBNs would be excessively activated to form neutrophil extracellular traps along with efficient payload release (small DP-SALs) and finally to induce excellent anti-RA effect.

Sevuparin Blocks Sickle Blood Cell Adhesion and Sickle-Leukocyte Rolling on Immobilized L-Selectin in a Dose Dependent Manner

Background: The cause and continuation of vaso-occlusion in sickle cell disease (SCD) are fueled by the sickle-Red Blood Cells interactions with multiple other cell populations, promoting inflammation, obstructing the vasculature, and injuring the endothelium, leading to broad manifestations that affect most vital organs. Recent studies have identified multiple cellular components and molecular factors that contribute to the pathophysiology of SCD as reviewed by Zhang et al 2016 in Blood. It is likely that a multi-targeted approach for addressing SCD vaso-occlusion will be required to achieve the best clinical outcome. Sevuparin (DF02), a novel drug in Phase 2 for acute treatment of vaso-occlusive crisis in SCD (NCT02515838), is a polysaccharide blocking abnormal adhesion and thereby normalizing obstructed blood flow. In vitro and in vivo studies have shown potent anti-adhesive effects with a multimodal mechanism of action. In this study, we evaluate the effects of sevuparin on the adhesion of sickle whole blood from individual patients to endothelial cells (HUVECs) and vascular cell adhesion molecule-1 (VCAM-1) (Flow Firm Adhesion) and sickle-leukocyte rolling adhesion on L-selectin (Flow Dynamic Adhesion) using a standardized microfluidic flow-based adhesion assay.Methods: Blood was obtained from homozygous SCD patients (n = 12, age range 15-25yrs) in sodium citrate after obtaining informed consent. A comprehensive assessment of the effect of sevuparin on whole blood adhesive properties during simulated blood flow was assessed using standardized Flow Firm Adhesion and Flow Dynamic Adhesion assays (Functional Fluidics, Detroit MI). Flow Firm Adhesion: Whole blood firm adhesion was measured during physiologic flow in microfluidic channels (Fluxion-Bioflux 1000, San Francisco, CA) coated with either VCAM-1 or cultured HUVECs. HUVECs were activated by TNF-alpha (25ng/mL x 24 hrs.) and Histamine (100mM x 10min) prior to the assay. Whole blood was treated with increasing doses of sevuparin (0, 3, 7, 21, 200µg/mL) for 30 min. Dose response of whole blood adhesion index (cells/mm2) to sevuparin was measured. Flow Dynamic Adhesion: Rolling adhesion of isolated sickle-leukocytes on an L-selectin coated microfluidic channel was measured during physiologic flow. Isolated sickle-leukocytes were treated with increasing doses of sevuparin (0, 3, 7, 21, 200µg/mL) for 30 min. Dose response of rolling cell density (cells/mm2), rolling cell percentage (%), and average rolling velocity (µm/s) to sevuparin was assessed. Cell identification and tracking of rolling were digitally analyzed.Results:Statistically significant inhibition of sickle whole blood adhesion to HUVECs was observed at 3.0 µg/mL of sevuparin (p<0.001). In the same manner, statistically significant inhibition of sickle whole blood adhesion to VCAM-1 was observed at 200 µg/mL of sevuparin (p=0.033, absolute adhesion, p=0.001, % baseline adhesion). Each patient sample demonstrated a reduction in adhesion. Sevuparin also demonstrated a statistically significant dose-dependent reduction of sickle leukocyte rolling cell density (cells/mm2), rolling cell percentage (%), and an increase in average rolling velocity (µm/s) on L-selectin. Patient-to-patient variability in sevuparin response was observed.Conclusions: Sevuparin blocks both sickle whole blood and isolated sickle-leukocyte adhesive interactions under physiologic flow at clinically relevant concentrations. The blocking of adhesion to VCAM-1 indicates that sevuparin acts in the same manner as other heparinoids in vitro, and block the interaction with VLA-4. L-selectin is another possible target for sevuparin therapy now confirmed at the cellular level. Clinically, Okpala et al 2002 has shown that L-selectin expression by monocytes is increased in vaso-occlusive crises, compared to steady state and that both mononuclear cell and neutrophil L-selectin expression is also higher in patients with certain complications of SCD. Here we show that sevuparin acts in a multicellular manner, blocking both SS-RBC firm adhesion and L-selectin-mediated rolling adhesion of sickle-leukocytes, as well as functionally interacting with yet another key adhesion receptor VCAM-1. This further adds to sevuparin's multimodal action and its potential clinical benefits in treating the complex mechanisms manifested in vaso-occlusion and complications in SCD. DisclosuresLindgren:Dilaforette AB: Employment. White:Functional Fluidics: Employment, Equity Ownership. Liu:Functional Fluidics: Employment, Equity Ownership. Jendeberg:Dilaforette AB: Employment. Hines:Fucntional Fluidics: Employment, Equity Ownership.

Plane of nutrition during the preweaning period but not the grower phase influences the neutrophil activity of Holstein calves

Two experiments were conducted to determine the influence of plane of nutrition during (1) the pre- and immediate postweaning periods and (2) the grower phase on the performance and leukocyte responses of Holstein calves. In experiment 1, 39 (2±1d old) colostrum-fed heifer calves were randomly assigned to 2 planes of nutrition, a low (LPN; n=19) and a high plane of nutrition (HPN; n=20). Calves in the LPN treatment were offered 418g/d of dry matter (DM) of a 20% crude protein (CP)/20% fat milk replacer, whereas calves in the HPN treatment were offered 747 and 1,010g/d of DM of a 28% CP/20% fat milk replacer during wk 1 and wk 2 to 6, respectively. Calves were offered ad libitum access to a calf starter until the end of the study. Peripheral blood samples were collected on d 3, 10, 21, 45, 47, 53, and 91 for many ex vivo leukocyte responses and biochemical analyses. A nutrition × time interaction was present for average daily gain and feed efficiency (feed:gain ratio). A nutrition × time interaction existed for plasma glucose concentration. Neutrophil L-selectin expression was greater in calves fed the LPN than HPN on d 3 and 21. The percentage of neutrophils producing an oxidative burst (OB) when cocultured with Escherichia coli tended to be greater and was greater in calves fed the LPN than HPN on d 10 and 21, respectively. In addition, neutrophils from calves fed the LPN had greater OB intensity throughout the neonatal period (0 to 21d). However, plasma haptoglobin was not different between the 2 planes of nutrition throughout the study. In experiment 2, 50 heifer calves that were all previously fed a HPN similar to that described for experiment 1 were randomly assigned to 2 planes of nutrition (HPN and LPN) during the grower phase (5 pens/treatment; 5 calves/pen). Calves fed the HPN during the grower phase were fed 4.1kg of concentrate DM (pellets; 22.4% CP, DM basis) per head per day, whereas calves on the LPN during the grower phase were fed 1.6kg of DM of the same concentrate per head per day. All calves were fed alfalfa hay (16.2% CP; DM basis) ad libitum. Overall, average daily gain was greater in HPN calves than LPN calves. No differences were noticed for concentrations of plasma urea nitrogen, glucose, neutrophil L-selectin expression, percentage of neutrophils producing OB, and plasma haptoglobin concentration between the 2 planes of nutrition. In summary, intake and performance were improved in calves fed the HPN than calves fed the LPN in both experiments. The neutrophil responses of calves fed an LPN were more active during the preweaning period than calves fed an HPN; however, this response was not observed during the immediate postweaning period or the grower phase.

50. Monoacylglycerol lipase inhibition reduces inflammation in a murine model of acute lung injury

Endocannabinoid signaling is terminated by enzymatic hydrolysis, a process that for 2-Arachidonoylglycerol (2-AG) is mediated by monoacylglycerol lipase (MAGL). The JZL184 (drug) inhibits MAGL with potency and selectivity. It increases 2-AG levels, an endocannabinoid that acts on CB1 and CB2 cannabinoid receptors. The aim of this work was to investigate the effects of MAGL inhibition in a murine model of acute lung injury (ALI) induced by LPS. Twenty C57BL/6 male mice were used (5–8/group); mice were divided into 3 groups, vehicle + saline (V), vehicle + LPS (L), and JZL184 + LPS (J). Mice were treated with JZL184 (16 mg/kg) or vehicle and after 60 min they received a LPS intranasal instillation (100 μg/mL; 1 μL/g) or sterile saline. Six hours later all mice were euthanized and blood, bone marrow and bronchoalveolar lavage fluid (BAL) were harvested. L group showed an increase in the leukocyte count in the BAL especially neutrophils; JZL184 treatment decreased these two parameters. Lymphocytes count in the blood were increased in L group and that treatment with JZL184 prevented these changes. JZL184 did not prevented the decrease of granulocytes number in bone marrow. JZL184 reduced vascular permeability. JZL184 also decreased β2-integrin and increased L-selectin expression in neutrophils in blood. JZL184 treatment decreased IL-6 and MCP-1 concentration in the BAL. ALI establishment is evident 6 h after LPS nasal instillation and that MAGL inhibition has anti-inflammatory effects in this ALI murine model.FAPESP2011/10181–7;2009/51886–3

Achyrocline satureioides(Lam.) D.C. Hydroalcoholic Extract Inhibits Neutrophil Functions Related to Innate Host Defense

Achyrocline satureioides (Lam.) D.C. is a herb native to South America, and its inflorescences are popularly employed to treat inflammatory diseases. Here, the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences of A. satureioides on neutrophil trafficking into inflamed tissue were investigated. Male Wistar rats were orally treated with A. satureioides extract, and inflammation was induced one hour later by lipopolysaccharide injection into the subcutaneous tissue. The number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate, and adhesion molecule and toll-like receptor 4 (TLR-4) expressions and phorbol-myristate-acetate- (PMA-) stimulated oxidative burst were quantified in circulating neutrophils. Leukocyte-endothelial interactions were quantified in the mesentery tissue. Enzymes and tissue morphology of the liver and kidney were evaluated. Treatment with A. satureioides extract reduced neutrophil influx and secretion of leukotriene B4 and CINC-1 in the exudates, the number of rolling and adhered leukocytes in the mesentery postcapillary venules, neutrophil L-selectin, β2-integrin and TLR-4 expression, and oxidative burst, but did not cause an alteration in the morphology and activities of liver and kidney. Together, the data show that A. satureioides extract inhibits neutrophil functions related to the innate response and does not cause systemic toxicity.

The Activation Pattern of Blood Leukocytes in Head and Neck Squamous Cell Carcinoma Is Correlated to Survival

Head and neck squamous cell carcinoma (HNSCC) is known to cause substantial immunosuppression. The present study was designed to characterize blood leukocyte activation in HNSCC and to investigate if the individual activation pattern could be related to tumor progress and survival. The leukocyte activation profile of HNSCC patients and healthy controls was assessed with flow cytometry. HNSCC patients displayed increased numbers of monocytes, neutrophils and total leukocytes as well as an enhanced neutrophil/lymphocyte ratio. In addition, patients had a higher percentage of CD69+, CD71+ and CD98+ T cell subsets and NK cells, and a reduced expression of L-selectin in CD14highCD16+ monocytes and neutrophils, when compared to controls. These changes could be correlated to both tumor burden and spread to lymph nodes. Among the cancer patients an increased neutrophil/lymphocyte ratio, a low neutrophil and CD14high CD16+ monocyte activation state and an elevated CD4/CD8 ratio were related to poor survival. In contrast, a high percentage of CD98+ Th cells appeared to be associated with a better outcome. Taken together, the present data indicate that HNSCC causes activation of blood leukocytes and that the individual activation pattern can be linked to prognosis.

Technical note: Determination of preanalysis storage temperature and time allowances for ex vivo innate immune responses

Evaluation of ex vivo innate immune responses requires the transport of blood to a laboratory for analyses. Storage temperature and duration are highly variable in the published literature. The objective of the present study was to establish recommended storage temperature and duration for blood before analysis of several ex vivo innate immune responses in vitro. Peripheral blood was collected from 8 clinically healthy, 12-mo-old Holstein heifers and stored either on ice or at ambient temperature for 1.5, 3.5, 5.5, 7.5, 9.5, and 24h before conducting analyses for lipopolysaccharide stimulated whole blood tumor necrosis factor-α (TNF-α) secretion, neutrophil L-selectin expression, and neutrophil phagocytic and oxidative burst capacities when exposed to an Escherichia coli. L-Selectin expression was greater in neutrophils stored on ice as early as 1.5h after blood collection. In addition, when neutrophils were stored on ice, L-selectin expression at 24h was not different from that at 1.5h after blood collection. Measures of innate immune cell function, including whole blood TNF-α secretion and neutrophil phagocytic and oxidative burst abilities, were decreased in blood stored on ice. Both TNF-α secretion and neutrophil oxidative burst intensity did not differ at 24h compared with 1.5h after blood collection when blood was stored at ambient temperatures. These data indicate that both storage temperature and duration influence measures of innate immune responses. Therefore, storage durations should be minimized and storage protocols kept consistent to reduce the influences of storage conditions on experimental results.

Innate immune responses of temperamental and calm cattle after transportation

The objective was to investigate measures of cellular innate immune responses among calm and temperamental Brahman bulls in response to handling and transportation. Sixteen Brahman bulls (344 ± 37 d of age; 271.6 ± 45.5 kg BW) classified as either calm ( n = 8) or temperamental ( n = 8) were loaded onto a trailer, transported for 4 h to a novel facility, rested 16 h overnight, and then were returned to their original facility after a 4 h transport. Blood samples were collected immediately prior to (time 0) and at 24, 48, and 96 h after initial loading for analyses of innate immune and blood parameters. Leukocyte counts did not differ ( P > 0.05) due to temperament before or after transportation, but neutrophil:mononuclear cell ratios were greater in temperamental bulls compared to calm bulls at 24 h. At 24 h, expression of peripheral neutrophil β 2-integrin decreased among all bulls compared with 0 h ( P < 0.01). Temperamental bulls had greater glucose and cortisol than calm bulls ( P < 0.01) at 48 h; whereas calm bulls had elevated neutrophil l-selectin expression, and phagocytic and oxidative burst activity compared with temperamental bulls ( P < 0.10) at 48 h. The supernatant collected from endotoxin-stimulated whole blood cultures had greater TNF-α concentrations at 48 h than at the other time points ( P < 0.05), but no temperament effect was observed ( P > 0.05). In contrast, 96 h after initial loading the supernatant TNF-α concentrations were lower ( P < 0.05) among all cattle. Lastly, transportation increased neutrophil phagocytosis, oxidative burst, and cell adhesion molecule expression 96 h post-transportation and the effect was more pronounced among calm bulls. These data suggest that neutrophils from calm bulls are more likely to resist microbial invasion at 96 h after transportation than neutrophils from temperamental bulls.

Effects of changing milk replacer feedings from twice to once daily on Holstein calf innate immune responses before and after weaning

The objectives of this study were to determine the effects of switching Holstein calves to once-daily feeding during the fourth week of life (24±2.3 d of age; once-fed n=22; twice-fed n=22) on innate immune responses, and to evaluate whether carry-over effects occurred when the calves were weaned during the seventh week of life. Peripheral blood samples were taken immediately before the change in feeding strategy (24 d of age) and at 27, 31, 45, 48, 52, and 66 d of age and were analyzed for circulating cortisol, haptoglobin, total leukocyte counts, neutrophil:mononuclear cells, and hematocrit percentage. Heparinized whole blood was also stimulated with lipopolysaccharide (LPS) for 24h and the concentration of tumor necrosis factor-α (TNF-α) in the supernatant was analyzed. Neutrophil L-selectin and β2-integrin expression were analyzed by flow cytometry. Simultaneous neutrophil phagocytic and oxidative burst responses to a heat-killed Escherichia coli were quantified by dual-color flow-cytometry. Treatment (once-daily or twice daily feeding) had no effect on pre- or postweaning performance. Once-fed calves tended to have more circulating neutrophils at 27 d of age, greater expression of L-selectin on neutrophils at 31 and 45 d of age, and greater intensity of phagocytosis at 45 d of age. Once-fed calves secreted less TNF-α in LPS-stimulated whole blood cultures at 45 d of age compared with twice-fed calves and this tended to persist through the immediate postweaning period. None of the other immune parameters differed after weaning between the preweaning feeding strategies. Consolidating calf milk replacer into one feeding during the fourth week of life was likely a mild and acute stressor, as evidenced by transient neutrophilia in the absence of suppressed functional capacities of neutrophils. Future research should address the mechanism and immunological significance of the persistent decreased TNF-α response in once-fed calves.

2-Aminoethoxydiphenyl borate (2-APB) reduces respiratory burst, MMP-9 release and CD11b expression, and increases l-selectin shedding in bovine neutrophils

This study describes the effect of 2-aminoethoxydiphenyl borate (2-APB), a putative store-operated calcium (Ca 2+) entry (SOCE) inhibitor, on reactive oxygen species (ROS) production, matrix metalloproteinase 9 (MMP-9) release, CD11b and l-selectin (CD62L) expression, size changes and apoptosis in bovine neutrophils stimulated with platelet-activating factor (PAF). It was observed that doses ⩾1 μM 2-APB significantly reduced ROS production, whereas 50 and 100 μM 2-APB reduced MMP-9 release induced by PAF. Moreover, concentrations ⩾10 μM 2-APB reduced CD11b expression and increased l-selectin shedding. PAF induced size changes in neutrophils, and this effect was inhibited by 2-APB. From this work it is possible to conclude that 2-APB at concentrations that inhibit SOCE responses was able to inhibit ROS and MMP-9 release and CD11b expression, and increase l-selectin shedding, suggesting that the Ca 2+ channel involved in SOCE is a potential target for the development of new anti-inflammatory drugs in cattle.

Alterations of neutrophil l-selectin and CD18 expression by tobacco smoke: implications for periodontal diseases

Alterations in neutrophil functions by both chronic low levels of tobacco and by acute short-term higher levels of tobacco smoke, as encountered during the act of smoking, may play a role in the pathogenesis of periodontal diseases in smokers. Among the early migration events of neutrophil function is the alteration in surface expression of L-selectin and the CD11/18 integrins. In the present study we examined the effect of in vitro smoke exposure and nicotine alone on the expression of these 2 adhesion molecules in neutrophils from smokers and non-smokers. We also determined the physiological relevance of this in vitro system by assessing the levels of nicotine exposure in this in vitro system and comparing these levels to acute and chronic levels of nicotine in saliva and gingival crevicular fluid. Peripheral neutrophils were isolated from the blood of smokers (< 1 pack/d) and non-smokers and incubated in vitro with either cigarette smoke (0-5 min), 10−7 M F-met-leu-phe, or nicotine alone at 1.62 mg/ml to 162 ng/ml (10−2 M-10−6 M). The neutrophils were then incubated with fluoresceine conjugated anti-Leu8 (L-selectin), anti-CD 18 (CD 18 integrin), or γ-4 (non-specific control), fixed and analyzed by flow cytometry. With cigarette smoke exposure, there was an approximate 75% shedding of L-selectin in both smokers and non-smokers with no marked difference between groups at 1-5 min of smoke exposure. Cigarette smoke exposure resulted in a 15-20% increase in CD18 expression in both smokers and non-smokers. At all time points, there was slightly greater but statistically insignificant expression of CD 18 integrin in non-smokers when compared to smokers. These patterns of CD 18 increases and L-selectin shedding were similar in magnitude to incubations with 10−7 M F-met-leu-phe. Acute smoke exposure resulted in elevation of nicotine in the smoke box to 529 ng/ml at 5 min, in saliva from 109.2 ng/ml before smoking to 1821.4 ng/ml after smoking, and in gingival crevicular fluid to 5961 ng/ml after smoking. No significant alterations in L-selectin or CD 18 expression were noted with in vitro nicotine from 1.62 mg/ml to 162 ng/ml.

Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital microscopic studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression of l-selectin and β2-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to fMLP, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation.

Ethanol alters neutrophil membrane mechanics and adhesion molecule expression

Ethanol (EtOH) has been linked to both protective and damaging effects in inflammatory and immune responses of leukocytes. To characterize EtOH effect on neutrophil mechanics, we studied neutrophil rolling, tethering, and firm arrest behavior at physiologically relevant EtOH concentrations achieved in humans by alcohol consumption (&lt;0.5 v/v%). Neutrophils were perfused over parallel‐plate flow chambers with P‐selectin‐coated beads or confluent HAEC (human aortic endothelial cells) monolayer at a venous shear rate of 100 s−1. EtOH decreased the adhesion efficiency of neutrophils from 0.085 to 0.031, increased the average tether length and tether growth velocity fourfold, and decreased bond forces, suggesting a lower degree of membrane‐cytoskeleton association. When perfused over IL‐1‐activated HAEC, the fraction of neutrophils converting to firm arrest decreased from 36% to 24% with EtOH, and average rolling length and rolling velocity both increased with EtOH (24.8 to 33.7 μm and 2.5 to 3.4 m/sec, respectively). EtOH resulted in ~20% increase in L‐selectin and PSGL‐1 expression in quiescent neutrophils as measured by flow cytometry, and significantly inhibited the upregulation of CD11b and shedding of L‐selectin in fMLP‐activated neutrophils. Our results indicate that EtOH affects bond mechanics and surface molecules to change neutrophil response. This work was supported by NIH R01 HL56621.

Endogenous glucocorticoids control neutrophil mobilization from bone marrow to blood and tissues in non-inflammatory conditions.

We have shown that endogenous glucocorticoids control neutrophil mobilization in the absence of inflammation. In this study the role of the glucocorticoid receptor (GR) in the physiological control of neutrophil mobilization was investigated, focusing on the specific mechanisms for mature neutrophils in bone marrow, circulating neutrophils and endothelial cells. Male Wistar rats were treated with RU 38486 or adrenalectomized. Cell numbers in bone marrow and circulation were morphologically quantified and expressions of L-selectin determined by flow cytometry. Expressions of P-selectin, E-selectin, PECAM-1, VCAM-1 and ICAM-1 were measured by immunohistochemistry on vessels of cremaster muscle and their mRNA levels quantified in primary cultured endothelial cells. NF-kappaB activity in neutrophils and endothelium was quantified by EMSA. RU 38486 treatment altered the maturation phases of neutrophilic lineage and reduced expression of L-selectin in mature neutrophils from bone marrow; increased the number of neutrophils in the circulation and elevated the expression of L-selectin in these cells. P-selectin and E-selectin expression in endothelial cells was unchanged by adrenalectomy or RU 38486 treatment. Membrane expressions, mRNA levels of ICAM-1, VCAM-1 and PECAM-1 and NF-kappaB translocation into the nucleus were higher in the endothelium of adrenalectomized and RU 38486 treated rats. Endogenous glucocorticoids, through activation of GR on neutrophils, physiologically control the rolling behaviour of these cells and, by modulating endothelial functions, affect their adhesiveness. The molecular mechanism induced by activated GR is different in each cell, as NF-kappaB translocation was only altered in endothelial cells.

Synthetic inotropes inhibit the expression of adhesion molecules and augment the expression of l-selectin in polymorphonuclear neutrophils

To elucidate differential functional and phenotypic changes in response to clinically relevant synthetic inotropes plus the generation of oxidative free radicals by polymorphonuclear neutrophils (PMN), and changes in the expression of L-selectin and Mac-1 on the surface of PMN were examined in the presence of dobutamine and dopexamine in pharmacological concentrations. Prospective, in vitro study. Research laboratory. Human PMN obtained from healthy donors. PMN were pretreated with dobutamine 147.99 nM or 147,990 nM, or dopexamine 100 nM or 100,000 nM, followed by stimulation with FMLP. Stimulated neutrophils were incubated with antibiodies against CD11b or CD62l and assessed by flow cytometry. Additional probes were assessed by flow cytometry for the generation of oxidative free radicals. Low concentrations of both synthetic inotropes significantly inhibit the suppression of CD62l expression following stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine; high concentrations antagonize this effect. High concentrations of both synthetic inotropes suppresses the expression of CD11b. Neither dobutamine nor dopexamine modified the generation of oxidative free radicals. While the upregulation of Mac-1 expression is inhibited in a dose-dependent manner, the expression of L-selectin is enhanced at low concentrations of dobutamine and dopexamine and partly counter-regulated at high concentrations. It seems that synthetic inotropes can modulate the immunomodulatory ability by inhibition of PMN rolling and modification of PMN adherence and diapedese.

NAD(P)H oxidase: where there's smoke, there's fire

in industrialized nations such as the United States, tobacco represents the leading cause of deaths, with a majority of these being associated with cardiovascular disease, rather than cancer ([2][1], [20][2]). The vasculature develops an inflammatory and thrombogenic phenotype in response to many

Activated Protein C in the Cardioplegic Solution on a Porcine Model of Coronary Ischemia-Reperfusion has Deleterious Hemodynamic Effects

In reperfusion injury activation of coagulation and inflammation contribute to organ dysfunction. Activated protein C (APC) exhibits anticoagulant and anti-inflammatory properties in models of reperfusion injury. We hypothesized that APC could be cardioprotective after ischemia and cardiopulmonary bypass (CPB). 20 pigs, undergoing 120 min of CPB and aortic cross-clamping, were randomized to receive 1 mg of human APC or placebo to the last cardioplegic solution given 15 min before declamping to the systemic circulation. After aortic declamping the heart was supported by continuing CPB for 30 min followed by 30 min surveillance. Thrombin-antithrombin complexes, neutrophil L-selectin expression in blood and myeloperoxidase activity (MPO) of myocardial biopsies were measured. There was no indication of APC-induced increased bleeding. Thrombin levels were significantly lower in the APC group than in the placebo group and so were the rates of thrombin formation during the first 3 min of reperfusion and between 10 and 30 min after declamping. There were no differences in MPO or in the proportion of L-selectin (+) to L-selectin (-) neutrophils between groups. Significant systolic hypotension in the APC group was observed at 30 and 45 min compared with the placebo group which associated with the increased mortality observed in the APC group (p = 0.019). Human APC in cardioplegic solution during CPB in pigs, decreased reperfusion induced thrombin formation with no associated bleeding. No anti-inflammatory effects of human APC were seen. However, in this setting, APC caused hemodynamic deterioration. The observed phenomenon could be explained by systolic hypotension potentially produced by the release of vasoactive substances generated by the APC activation of PARs in the endothelium.

Activation of neutrophils and monocytes by a leukocyte-depleting filter used throughout cardiopulmonary bypass

Cardiopulmonary bypass elicits systemic inflammation. Depletion of circulating leukocytes might alleviate inflammatory response. We studied the effects of a leukocyte-depleting filter on phagocyte activation during cardiopulmonary bypass. Fifty patients undergoing coronary artery bypass grafting were randomly allocated into an arterial line leukocyte filter group (n = 25) with a Pall LeukoGuard 6 leukocyte-depleting filter (LG6; Pall Biomedical, Portsmouth, United Kingdom) and a control group without any filter (n = 25). Blood sampling took place from arterial line at predetermined time points. In the filter group, the sample was taken immediately before the filter; to evaluate activation at the site, an additional sample was taken immediately after the filter. CD11b/CD18 and L-selectin expressions and basal production of hydrogen peroxide were determined with whole-blood flow cytometry, and plasma lactoferrin level was determined with enzyme-linked immunosorbent assay. Neutrophil CD11b expression was higher in the filter group than in the control group (P < .001). Likewise, monocyte CD11b expression, neutrophil hydrogen peroxide production, and lactoferrin plasma levels were all significantly higher, whereas neutrophil and monocyte counts and neutrophil L-selectin expression were all significantly lower in the filter group (all P < .001). At 5 minutes of CPB, CD11b expression increased across the filter on neutrophils (median difference 197 relative fluorescence units, range 45-431 relative fluorescence units, P < .001) and monocytes (median difference 26 relative fluorescence units, range -68-111 relative fluorescence units, P < .001). The LG6 arterial line leukocyte filter is ineffective in its principal task of diminishing phagocyte activation during cardiopulmonary bypass.

Effect of proinflammatory mediators and glucocorticoids on L-selectin expression in peripheral blood neutrophils from dairy cows in various stages of lactation

To determine whether proinflammatory mediators and glucocorticoids affect CD62L(L-selectin) expression on peripheral blood neutrophils from cows in various stages of lactation. 100 healthy dairy cows during early (13.1 +/- 0.79 days after parturition; n = 31), peak (58.7 +/- 1.64 days after parturition; 31), and mid (137.2 +/- 2.59 days after parturition; 38) lactation. In vitro effects of relevant proinflammatory mediators that are released in response to mastitis caused by gram-negative bacteria such as lipopolysaccharide (endotoxin), tumor necrosis factor-alpha, and platelet-activating factor (PAF) on CD62L expression on bovine neutrophils were assessed by flow cytometry. Influences of cortisol and dexamethasone on CD62L expression on bovine neutrophils were also investigated. Basal CD62L expression on neutrophils from cows during early, peak, and mid lactation were similar. Lipopolysaccharide and tumor necrosis factor-alpha had no effect on CD62L expression on neutrophils from cows at any stage of lactation. Conversely, PAF elicited a time- and dose-dependent, down regulatory effect on CD62L expression. However, no differential shedding of CD62L from neutrophils of cows at any stage of lactation were detected. In addition, no effects on CD62L expression on bovine neutrophils after whole blood incubation with cortisol or dexamethasone were observed. Incubation with glucocorticoids did not prevent the down regulatory effect of PAF on CD62L expression. Comparable basal CD62L expression on bovine neutrophils and equal amounts of CD62L shedding from bovine neutrophils during all stages of lactation suggest that variations in CD62L density are not a likely cause of susceptibility of cows to coliform-induced mastitis during early lactation.

454. Evaluation of Polyethylene Glycol (PEG) Modification of Adenoviral Vectors To Reduce Innate Immune Response and Increase Vector Persistence

Gene therapy is a therapeutic strategy that may potentially treat a multitude of clinically important conditions. To these ends, adenovirus vectors (Ad-vectors) are a promising means to deliver specific genes of interest to target cells. The efficacy of Ad-vectors is limited by the innate immune response against these infectious particles. Following transduction in vivo, Ad-vectors activate the innate immune system resulting in rapid vector clearance and acute inflammation of transduced tissues. The infiltration of effector leukocytes, such as neutrophils, is a hallmark of the innate immune response and may play a major role in Ad-vector clearance. The aims of this work were to explore neutrophil-Ad-vector interactions and to identify the essential molecules mediating this interaction. C57/B6 mice were administered a Cy2-labeled first generation Ad-vector, AdLuc, intravenously and sacrificed at 3 hours. Neutrophil populations were isolated from the liver and analyzed by flow-cytometry for fluorescence. Compared to vehicle, neutrophils from Cy2-AdLuc treated mice demonstrated significant vector uptake confirming an important role for these innate cells in the host response to Ad-vectors. To further study Ad-vector neutrophil interactions, purified populations of human peripheral blood neutrophils were incubated with Cy2-AdLuc and analyzed using fluorescence microscopy and flow cytometry. Ad-vector uptake by neutrophils increased significantly over 20-60 minutes. Neutrophil uptake of Ad-vectors did not require viral capsid RGD motifs since internalization of the RGD-deleted Ad-vector, AdL.PB over 20-60 minutes was similar to AdLuc. Neutrophil pre-incubation with a blocking anti-CD11b mAb (ICRF44), but not control IgG, inhibited Ad-vector uptake demonstrating that this β2-integrin is required for Ad-vector internalization. To determine activation, neutrophil L-selectin expression was determined by flow cytometry following incubation with AdLuc. L-selectin expression, decreased significantly over 60 minutes following incubation with AdLuc consistent with neutrophil activation. These data confirm that neutrophils interact significantly with and are activated by Ad-vectors. Furthermore, neutrophil-expressed CD11b, but not viral capsid RGD motifs, is essential for adenoviral uptake. Future studies will focus on elucidating the mechanism of Ad-vector-mediated neutrophil activation and its role in vector clearance in vivo.